NOVEL PROTEIN COMPONENT AND ENCODING NUCLEIC ACID

Abstract: The present invention relates to novel protein as a component of bone marrow cells, blood cells or osteoblasts, or any variant or functional analogue of said peptide. The present peptides, sometimes denoted RP59 peptides, may assist isolation of mesenchymal stem cells for use on implant materials or in areas of degenerative tissue loss, for example in arthritic joints or in the context of periodontal disease. Regeneration after resective bone surgery or after bone damages may also be favoured via the supply of mesenchymal stem or progenitor cells. In addition, RP59 protein may help the assembly of tissue or tissue fragments <u>in vitro</u>, as intended for tissue engineering or regeneration. Diagnosis of bone, bone marrow or blood diseases and diseases may be helped by probes detecting RP59 protein or coding nucleic acid according to the invention. The invention also relates to expression vectors that comprise sequence coding for RP59, to organisms containing said expression vector, to methods for producing the polypeptide, and to methods for treatment and diagnosis of bone, bone marrow or blood disorders.

Description: NOVEL PROTEIN COMPONENT AND ENCODING NUCLEIC ACID Technical field The present invention relates to a novel protein component of bone marrow, the encoding nucleic acid as well as to various advantageous uses thereof. Background Bone marrow contains mesenchymal stem cells, which represent precursor cells for many tissues, among others for blood and bone (Pittenger, M.F., Mackay, A.M., Beck, S.C., et al., (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147; Nevo, Z., Robinson, D., Horowitz, S., Ha- sharoni, A. and Yayon, A. (1998), The manipulated mesenchymal stem cells in regenerated skeletal tissues, Cell Transplantation 7, 63-70). Several lines of evidence suggest that osteoblast precursor cells are transported by the blood stream to the regions of bone formation (Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276, 71-74). In vitro culturing of bone marrow cells may lead to the proliferation of blood forming cells in semisolid medium (Stanley, E.R. (1981) Colony Stimulating Factors In: The Lymphokines, J.W. Hadden and W.E. Stanley, eds. Humana Press, Clifton, ch. 6, pp 101-132), and to the formation of bone and other tissues when cells adsorb to plastic (Frie- denstein A.J. (1972) Determined and inducible osteogenic precursor cells Ciba Found. Symp. 11, 170-185, Bruder, S.P., Jaiswal, N., Ricalton, N.S. et al. (1998) Mesenchymal stem cells in osteobiology and applied bone regeneration Clin. Or- thoped. & Rel. Res. 355 (Suppl), S247-S256). In vivo, blood cells develop in marrow and other hematopoietic organs. For solid tissue forming cells, any differentiation traits or commitments of marrow cells are only prospective. Differentiation into productive connective tissue forming cells obviously cannot be realized before the cells have found their location in the tissue. Thus, the cells' differentiation be- haviour is associated to specific cellular contacts (Boudreau, N. and Bissell, M.J. (1998) Extracellular matrix signalling: integration of form and function in normal and malignant cells Curr. Op. Cell Biol. 10, 640-646 ). For example during differentiation of osteoblastic cells in rats, a sequence of stages has been defined via the expression of RNA species coding for fibrillar collagens. Here, the produced type of collagen RNA is specific for the position of the cell in the tissue (Wurtz, T., Ellerstrom, C, Lundmark, C. and Christersson, C. (1998) Collagen mRNA Expression during Tissue Segregation: the Temperospacial Order Coordinates Bone Morphogenesis with Collagen Fiber Formation Matrix Biology 17, 349-360, Marks, S.C. Jr, Lundmark, C, Wurtz, T., Odgren, P., Mackay, CA. Mason-Savas, A. and Popoff, S.N. (1999) Facial development and Collagen III RNA expression: Concurrent repression in the osteopetrotic (toothless, tl) rat and rescue after treatment with Colony-Stimulating Factor- 1 Dev. Dynam. 215, 117-125). Marrow represents a major source for mesenchymal stem cells in adult humans (Pittenger, M.F., Mackay, A.M., Beck, S.C, et al, (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147, Nevo, Z., Robinson, D., Horowitz, S., Hasharoni, A. and Yayon, A. (1998) The manipulated mesenchymal stem cells in regenerated skeletal tissues Cell Transplantation 7, 63-70), which also implicates that damaged connective tissue recruits commited stem cells via the blood stream for regeneration. To this end, medullary mesenchymal stem cells must, firstly, receive instructions to leave their environment for the vascular system, and secondly, to home into regenerative tissue compartments. These events require the destruction and re-establishment of cell attachments to surrounding structures as well as a coordinated regulation of cell proliferation. However, the principles according to which cells are attached and organized in bone marrow are poorly understood. Regulation of cell proliferation and differentiation in marrow probably requires, as anywhere else, the segregation of cell types as well as the mutual stimulation of cells. These events are influenced by cell-cell contacts and by contacts between cells and extracellular matrix. For bone marrow, extracellular material has been described as reticulin fibers containing collagen type III (Frisch, B. and Bartl, R (1990) Atlas of bone marrow pathology, Current Histopathology Vol 15, G.A. Gresham, ed., Kluwer Academic Publishers, Dordrecht, Ch. 7, pp 93-124). Pathological increase of reticulate fibers is referred to as myelofibrosis, accumulation of various other proteins as gelatinous transformation or amyloidosis (Frisch, B. and Bartl, R (1990) Atlas of bone marrow pathology, Current Histopathology Vol 15, G.A. Gresham, ed., Kluwer Academic Publishers, Dordrecht, Ch. 4, pp 57-63). Cues for the habitation of stem cells in target tissues are even less clear than the organization of marrow. It has however been observed that, for medical application, bone marrow transplantation is a more efficient regenerative aid than addition of cultivated stem cells (Nevo, Z., Robinson, D., Horowitz, S., Hasharoni, A. and Yayon, A. (1998) The manipulated mesenchymal stem cells in regenerated skeletal tissues Cell Transplantation 7, 63-70, Bruder, S.P., Jaiswal, N., Ricalton, N.S. et al. (1998) Mesenchymal stem cells in osteobiol- ogy and applied bone regeneration Clin. Orthoped. & Rel. Res. 355 (Suppl), S247-S256). This may suggest that marrow cells possess properties that favour habitation in different tissues, and are lost upon in vitro culturing. So far, an effect of cellular components cannot be assessed, since there is no experimental system that allows to test cell homing functions. Accordingly, there is a strong need within this field of further information and characterization of cellular components. Summary of the invention The present invention discloses an isolated predominant protein component originating from the bone marrow of rats that is present in embryonal cells, primitive erythroid cells and osteoblasts. The protein, called RP59, and its coding RNA are useful for the identification and the preparative isolation of such cells. Firstly, a DNA sequence is disclosed, which codes for a novel type of amino acid repeat structure. Consensus sequences define the essential elements of the repetitive units. Secondly, proteins containing these repeat structures are identified and located to cells of bone marrow, spleen, embryonic structures and to osteoblasts. The invention also relates to expression systems for production of RP59, pharmaceutical applications of said peptides and nucleic acids as well as compounds that are capable of specifically recognising the present nucleic acids or encoded peptides. Brief description of the drawings Fig. 1 shows in situ hybridisation with RP59 probe of a histological section through the femur of a 5-day-old rat, displaying the region of the growth plate. Fig. 2 shows a denaturing agarose gel electrophoresis of polyA enriched RNA from tissues of 5-day-old and fetal rats, transferred to filter membrane and hybridised to RP59 probe. Fig. 3 shows an SDS gel electrophoresis of protein extracts from femora of 5-day- old rats and detection of RP59 protein. Fig. 4 shows tissue sections through a rat embryo, stained for all tissues (HTX), RP59 RNA (ISH) or RP59 protein (IHC). Fig. 5 shows appearance of RP59 RNA in day 9-embryos (A), and RP59 protein in adult spleen (B) and bone bone marrow (C). Definitions The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term "functional fragment" relates to any fragment capable of uses similar or equivalent to the uses of the protein according to the invention, such as the identification and the preparative isolation of embryonal cells, primitive erythroid cells and osteoblasts. Functional fragments are further discussed and examplified below in the detailed description of the invention. Similarly, the term "analogue" of the present protein also relates to molecules that exhibit similar or equivalent properties as the present protein, e.g. the ones mentioned above. "Subsequence" refers to a sequence of nucleic acids or amino acids that comprises a part of a longer sequence of nucleic acids or amino acids (e.g., polypeptide), respectively, and is intended to include variations in the sequence such as substitution, insertion, addition and re-arrangements of nucleotides or amino acids, which do not have substantial adverse effect on the nucleic acid or the encoded polypeptide. The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides. The term "consensus sequence" means a sequence which is highly conserved between several regions. The phrase "hybridising specifically to" refers to the binding, duplexing, or hybridising of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will hybridise to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point Tm for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the molecules with complementary sequences are in double-stranded form at equilibrium. If the target sequences are present in excess, at Tm, 50% of the probes are occupied. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. "Bind(s) substantially" refers to complementary hybridisation between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accomodated by reducing the stringency of the hybridisation media to achieve the desired detection of the target polynucleotide sequence. The term "antibody" refers to a polypeptide substantially encoded by an immu- noglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. "Formative cells" relates to cells that synthesize and secrete components of extracellular matrices and thus contribute to the formation of connective tissue. "Tissue engineering" combines the principles and methods of life sciences with those of engineering to elucidate fundamental understanding of structure-function relationship in normal and diseased tissue, to develop materials and methods to repair damaged or diseased tissues, and to create entire tissue replacements (Langer, R. and Vacanti, J.P. (1993) Tissue engineering. Science 260, 920-926). The control of cell responses to implant material, the manipulation of the healing environment and the production of cells and tissues for transplantation into the body are thus included (Hubbell, J.A. (1995) Biomaterials in tissue engineering. Biotechnology 13, 565-576). "RP59" refers to protein containing one or more of the amino acid sequences according to the invention as well as to such proteins that are modified by glycosyla- tion, phosphorylation, carboxylation or by other prostetic groups. Thus, the term "RP59" is herein used interchangeably with the term protein/polypeptide according to the invention. Detailed description of the invention In a first aspect, the present invention relates to an isolated protein comprising all or parts of the amino acid sequence according to SEQ ID NO: 2, or a functional fragment or an analogue thereof, which is suitable for identification and isolation of progenitors to bone forming cells or hematopoietic cells. In the present context, the term "isolation" should be construed as "isolated from its natural surroundings". Preferably, the protein comprises essentially all of said sequence, even though it is to be understood that any fragment, such as a peptide or polypeptide, that exhibits the herein described function, or substantially the same function, is is also encompassed. In the present application, the term "comprising" should be construed as "including, but not limited to". Further, the term "essentially all of the sequence" means at least about 70%, such as at least about 80%, preferably at least about 90%) and most preferably at least about 95% of the sequence. In specific embodiments, the present invention relates to functional fragments of the protein. Thus, one embodiment is a fragment which comprises the artificial consensus sequence (I): C-Q-E-(a)-T-V-P-Q-X-(b)-S-(c)-K-(a) (I) wherein X denotes any amino acid; (a) denotes T or I, preferably T; (b) denotes H or Y; (c) denotes P or S. Another embodiment is a fragment which comprises the artificial consensus sequence (II): (d)-Q-D-M-A-(e)-P-E-A-L-S-P-K-(f) (II) wherein (d) denotes C, P or H; (e) denotes G or E; and (f) denotes M or T. A further embodiment is a fragment which comprises the artificial consensus sequence (III): L-S-H-K-T-X-(g)-E-X-X-(h)-P-(i)-X (III) wherein X denotes any amino acid; (g) denotes Q or K; (h) denotes E or G; and (i) denotes E or K. In the formulas given above, it is to be understood that X may be any amino acid, with the only requirement that it does not interfere with the function of the fragment. One particularly advantageous embodiment of a fragment according to the invention is a peptide, which comprises the sequence defined by amino acid nos.262- 281 as defined in SEQ ID NO:2. This specific peptide is discussed in further detail below in the section "Experimental". In further embodiments, the invention relates to polypeptides comprising two or more, such as three, four, five etc., of the above disclosed fragments as repetitive elements. Such polypeptides may be preferred in applications where it is desired to enhance the function of one fragment. Thus, polypeptides of any lengths are included within the scope of the present invention, provided they are suitable for identification and isolation of progenitor cells for bone and blood. As mentioned above, SEQ ID NO:2 shows the peptide sequence derived from the open reading frame of the cDNA encoding it and illustrated in SEQ ID NO: l. The amino acid sequence of SEQ ID NO:2 is disclosed in further detail in Tables 1 and 2 below, wherein repetitive elements starting with residue 144 are arrayed in double repeats (residues 144 to 337) and single repeats (residues 379 to 449). Table 1 : Translation of open reading frame MDVRKDQSHLTLNQTPEAHVEKSHAPDIIGSWSLRNREQLKKRKAEAQGR QTSQWQLGEQKKRKYQRTGKGNRRGRKRQGNVEQKAESWSQTENERVQ EVLVPAEEETEYSGNTATQALPLRASPTKAVPTEHCSEVPQESLQ 144 CQEI-TIQNHS-QT HQRRDKAEALSPTT CQEIGVLQ-YSPKM CQDMAEPEARSPKM CQETAVPQTYSPKA HEDMAEYEALSPKM CRETPVPQYHSSKI PQDMTGPEALAPDM CQETTVSQNHSSKV PQDMAGPEALSLKM CQEPTVLQEHTLKI CQDVARPDVLSSKT HQEMTVPKALPCIT PGDAAGPEGCSPKT LPQSDVTPMSVTPG KNTSHPDLGTAVAE GCFSEAGECIVSED ISTKTHQEAVEPKF LSHKTYKEFTVPIV SSHKTIQESPGPEE YSPGSRHEMSEPED LSIKTCKNRDGPKH SLPE GAQEVG GAQRQDP DAQDIEDAGA FSQDLEE-482 Table 2: Consensus sequences of RP59 repeats X: no preference found Accordingly, the present invention relates to isolated proteins comprising one or more repetitive elements of at least about 10, such as about 14 amino acids, preferably arranged in double arrays. The present proteins occur naturally in fetal liver cells, spleen cells, cells of early embryonal stages and bone marrow cells as well as in osteoblasts. The proteins and polypeptides according to the invention are prepared by isolation from their natural sources, by synthetic methods or by gene re- combinant techniques. Methods for isolating proteins are well known to those skilled in this field. The preparation of the present protein and polypeptide by re- combinant technology will be disclosed in detail below (for a general method, see e.g. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 nd ed., Ed., Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989. For references regarding solid phase synthesis of polypeptides, see e.g. Stewart et al., Solid Phase Peptide Synthesis, 2 nd ed., Pierce Chem. Co., Rockford, 111 (1984). Thus, the present invention relates to all polypeptides, which are present in early embryos, spleen, fetal liver, bone marrow or bone and contain repetitive sequence elements as defined by the consensus sequences in Fig. 4b, which polypeptides are isolated from their natural environment. This includes peptides that include chemi- cal modifications, e.g. by carboxylation, glycosylation, lipididation or other prostethic groups. The invention also relates to analogues and variants, which does not contain the exact amino acid sequence depicted in Fig.4, but which are capable of acting in a competitive way with peptides containing these sequences, for instance in a cell binding assay. Similarly, homologuous sequences, i.e. RP59 sequences from other mammals than rat, are related. In a second aspect, the present invention relates to a nucleic acid encoding a polypeptide as described above. In a specific embodiment, the nucleic acid according to the invention comprises essentially all of the nucleotide sequence defined by SEQ ID NO: 1, or a variant of said sequence. More specifically, SEQ ID NO: l shows the sequence of RP59 cDNA, derived from several independent clones and RACE (rapid amplification of cDNA ends). Sequences were obtained after cycle sequencing in the presence of fluorescent terminators (ABI system), and alignment by the program seqman II (DNAstar). The start of longest possible translation product is illustrated by the start ATG defined as a miscellaneous feature located at base nos. 64-66 in SEQ ID NO: 1; while the corresponding stop codon is illustrated by the terminator TGA defined as a miscellaneous feature located at base nos. 1510-1512 of said sequence. Further, the present invention also relates to a nucleic acid, which is capable of hybridisation under stringent conditions with a sequence encoding the polypeptide or protein, as well as a nucleic acid which, but for the degeneracy of the genetic code, would hybridise to the above mentioned under the herein specified conditions. Thus, in the same way as for DNA, the present invention relates to RNA and any other macromulecules with a sequence of purine and pyrimidine bases, linked together by phosphodiester bonds, peptide bonds or other chemical conformations. The present molecules may be useful for antisense therapy and similar applications. Macromolecules disclosed here are ex- pected to hybridise to the nucleotide sequence of SEQ ID NO: l under stringent conditions, as defined above. The present nucleic acids are e.g. prepared by isolation from their natural source, such methods being well known to those of skill (see e.g. Laboratory Techniques in Biochemistry and Molecular Biology: Hybridisation With Nucleic Acid Probes, Part I); by polymerase chain reaction (PCR) (Innis et al. (1990) PCR Protcols, A guide to Methods and Application, Academic Press, Inc, San Diego) etc; chemical synthesis may e.g. be performed as a phosphotriester method (Narang et al., Meth. Enzymol. 68:90-99); a phosphodiester method (Brown et al., Meth. Enzymol. 68: 109-151); a diethylphosphoramidite method (Beaucage et al., Tetra. Lett., 22: 1859-1862 (1981); or a solid support method (US patent no. 4 458 066). A third aspect of the invention is an expression vector comprising the nucleic acid according to the invention as well as expression systems or casettes for expression of peptides according to the invention. Preferred expression vectors may be plas- mids, viruses, cosmids, autonomously replicating mini-chromosomes or elements which integrate into the genome of a host cell. In one embodiment, the vector includes the sequence according to the invention positioned downstream of a transcription promotor, and further expression control sequences, such as origin of replication, enhancer, and necessary processing information sites, such as ribo- some binding sites, RNA splice sites, polyadenylation sites and transcriptional terminator sequences. The present invention also relates to an organism, such as a cell or a microorganism, e.g. a bacterium or a yeast, as well as cells e.g. from insects, fungi, vertebrates and other multicellular organisms, capable of expressing the polypeptides and proteins according to the invention. Whole multicellular organisms like plants as expression mediators are included within the scope of invention. Included herein are also expression systems, wherein sequences of the invention are fused to other sequences, which may facilitate expression or isolation of the products, enhance the extent of expression or serve other purposes. (For general references to the production of fusion proteins, see e.g. Smith and Johnson (1988) Gene 67:31 ; Hopp et al. (1988) Biotechnology 6: 1204; La Vallie et al. (1993) Biotechnology 11 :187). The skilled in this field will easily choose the appropriate organism. A number of suitable host cell lines capable of secreting intact heterologous proteins and suitable to this end have been developed within this field and are easily available, e.g. COS cell lines, HeLa cells, etc. Accordingly, the present invention also relates to recombinant methods of producing the present polypeptides and proteins using the nucleic acids according to the invention, see e.g. Ausubel F.M. et al, eds (1998) Current Protocols in Molecular Biology, Ch.6, Screening of Recombinant DNA libraries. Thus, the present vectors are transferred into the host organism or cell line by well known methods, depending on the host chosen, e.g. by calcium chloride transfection in case of a procaryotic host or calcium phosphate treatment, electroporation etc. in case of a eucaryotic host. Once expressed, the peptides and proteins may be purified according to standard procedures of the art, such as HPLC purification, gel electrophoresis etc. (see e.g. Scopes, Protein Purification, Springer- Verlag, NY, 1982). In case of proteins, the skilled in this field recognizes that the protein produced may possess a conformation which differs from the desired, native conformation thereof. Thus, the present protein may need to be re-folded and methods of inducing re-folding are well known to those of skill, see e.g. Debinski et al (1993) J. Biol. Chem., 268:14065-14070. It is contemplated that RP59 proteins or peptides according to the invention are biologically active in the context of maintenance of mesenchymal stem cells in bone marrow, or during differentiation into bone or blood cells, or during translo- cation or attachment of stem cells at target tissues in the context of tissue regeneration. Thus, in a further aspect, the present invention relates to the present polypeptide and protein for use in the identification and isolation of progenitor cells for bone and blood. Such progenitor cells may proliferate in cell culturing systems and be used for reconstruction of bone tissue, supplement for blood cells, e.g. after irradiation or treatment with cytostatics, after surgery, or for treatment of perio- dontal diseases. Cells may be applied e.g. by injection or by direct spreading on tissue, eventually after surgical access. A composition according to the invention may also include pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents etc, e.g. sodium acetate, sodium chloride. Thus, the polypeptide according to the invention is useful in a medical context at treating and/or preventing and/or diagnosing bone and blood disorders, preferably to stimulate or engineer tissue regeneration, and accordingly the present invention also relates to methods of treatment wherein isolated progenitor cells are administered to a patient, in order to stimulate the regeneration and formation of bone or blood. Included herein are arthritic degeneration and disturbed reconstruction of bone tissue, diminished supply of blood cells, e.g. after irradiation or treatment with cytostatics, as well as bone loss, e.g. during periodontal diseases. The invention also relates to methods to aid regeneration of connective tissue after surgical resection, where isolated cells according to the invention may be administered to injured tissue surfaces. In addition, the present invention relates to the above defined nucleic acids for use as a medicament, the use thereof in the manufacture of a medicament, a pharmaceutical preparation comprising said nucleic acids together with a pharmaceutically acceptable carrier as well as methods for treatment involving the nucleic acids. The same general considerations and references discussed above in relation to the polypeptides will also apply for the therapeutic application of the present nucleic acids. In a further aspect, the invention relates to the use of isolated progenitor cells in the context of tissue engineering. Cells may be used to test responses to implant materials, for the development of materials and methods to the repair of damaged or diseased tissues, as well as for the creation of tissue replacements, which may comprise implants or artificial molds. Thus, the invention also relates to a method of preparing an implant or a transcutanous device suitable for use in transplantation surgery, more specifically, for the stimulation or induction of regenerative processes in bones or bone marrow, wherein a device according to the invention is used to improve the biocompatibility of said implant and/or transcutanous device, or a part thereof. Similarly, it is to be understood that the invention also relates to a method of improving the biocompatibility of an existing implant device by covering the surface thereof with isolated cells accordmg to the invention. The invention also relates to an implant or a transcutanous device per se, which is prepared, as a whole or in part, under the use of the products or by the method according to the invention. Another aspect of the present invention is a compound capable of specifically recognizing, preferably by specifically binding, a polypeptide according to the invention, which compound is a peptide or an antibody, preferably an antibody. The antibody may be monoclonal or polyclonal, for the preparation thereof, see e.g. Stites et al. (eds.) Basic and Clinical Immunology (4 ed.) Lange Medical Publications, Los Altos, CA; Coligan (1991) Current Protocols in Immunology, Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY). Consequently, the invention also encompasses a detection system comprising a compound or antibody as defined above, which recognises RP59 protein according to the invention in histological sections, Western blots, RIA, ELISA or other test systems, where the purpose of testing is the tracing of RP59 in analytical or diagnostic samples. These test systems may serve the analysis of RP59 function by scientific experiments or the medical diagnosis of RP59 presence in patients or in samples derived from patients. Thus, the present invention relates to use of a nucleic acid sequence or a compound as defined above, e.g. in the form of an antibody versus the present peptides, in diagnosis of disorders of bone, bone marrow or blood. In the most preferred embodiment, the nucleic acid is of human origin, while the compound is capable of recognising, e.g. by specific binding to, a human peptide. A last aspect of the present invention is a method of diagnosing, in vitro or in vivo, bone, bone marrow or blood disorders, wherein the nucleic acid and protein sequence according to the invention are used as diagnostic tools for the identification of pathologies in these tissues. Such diagnostic tools are intended to analyse the integrity of RP59 genes and to measure the expression of RP59 protein or nucleic acids coding for the RP59 or parts thereof. This comprises e.g. the analysis of patient DNA samples for genetic mutants or variants of RP59-specific sequences. For enhanced or reduced expression of RP59 RNA, in situ hybridisation of sections through tissue biopsies as well as hybridisation- or PCR-based assays testing iso- lated RNA from biopsies may be employed. Protein expression is monitored by immunohistochemistry and by analysis of extracts by ELISA or RIA. Detailed description of the drawings Fig. 1 shows in situ hybridisation of a histological section through the femur of a 5-day-old rat, displaying the region of the growth plate. Probes: anti-RP59 RNA sequences; anti collagen typ I RNA sequences; unrelated neomycine-resistance gene sequences (control). Arrows: osteoblasts. Cartilage with hypertrophied chon- drocytes is displayed at the top, bone marrow at the lower left corner. Fig.2 shows a denaturing agarose gel electrophoresis of poly-A enriched RNA from femur, heart, lung and spleen of 5-day-old rats and liver from day 17 embryos. RNA was loaded together with RNA fragments of standard lengths. Standard slot was cut off and stained with ethidium bromide. RNA was transferred to filter membrane and subjected to hybridisation with RP59 probe. Fig. 3 shows an SDS gel electrophoresis of protein extracts from femora of 5-day- old rats. Tissue pieces were extracted with 8M urea and maceration at room temperature; non-dissolved material was discarded. Extracts were mixed with reducing SDS buffer, boiled for 2 min, loaded on a discontinuous SDS gel along with standard proteins. Part of the gel was stained with Coomassie blue (std plus tot), part was electroblotted onto Immobilon membrane (Millipore) and incubated with antibody at a dilution of 1:1000 for the presence of RP59 (Western). Bound antibodies were revealed by an anti-antibody and phosphatase reaction (Daco, Copenhagen, Denmark). Similar filters probed with pre-immuneserum from the same rabbit gave no signal. Molecular mass of marker proteins in kD is indicated to the left. Fig. 4 shows paraffin sections through a 17-day-old rat embryo. HTX: hematoxy- lin-eosin stained section. ISH: In situ hybridization using RP59 probe, same enlargement as HTX. Signal is confined to the fetal liver. IHC: Immunohistochemis- try using anti-RP59 antibody, showing liver tissue in detail. Section was incubated with serum at a 1000X dilution, subsequently with goat anti rabbit serum coupled to horseradish peroxydase, and bound antibody was revealed with diaminobenzi- dine as substrate (Daco, Copenhagen, Denmark). Fig. 5 shows the presence of RP59 RNA and protein. A: section through a day-9 embryo after in situ hybridization. Arrow: amnion membrane. Positive cells observed mainly in the primitive streak. B: Immunohistochemistry of a section through adult spleen. Positive cells observed in the red pulp. C: Section through the growth plate of a femur of a 5-day old rat, showing strong signal in marrow cells (arrowheads), weak signal in osteoblasts (small arrows) and no signal in erythrocytes (big arrows). EXPERIMENTAL Below, the present invention will be described by way of examples, which are intended only as illustrating the invention and thus not limiting the scope as defined by the appended claims. All references used in the present application are hereby included herein by reference. Identification of RP59 mRNA RP59 sequences were initially identified in a cDNA library derived from rat femur, by screening individual mRNA sequences by in situ hybridisation. A cDNA library constructed in lambda ZAP (Stratagene) from rat femur-mRNA (Liao, H., Brand- sten, C, Lundmark, C. Christersson, C. and Wurtz, T. (1998) Osteonectin RNA and Collagen αl(I) RNA in the Developing Rat Maxilla Eur. J. Oral Sci. 106 (suppl 1), 418-423) was screened by plaque-hybridisation to DIG-labelled total cDNA from femur (Ausubel F.M. et al, eds (1998) Current Protocols in Molecular Biology, Ch.6, Screening of Recombinant DNA libraries). An excess of unlabelled rat cotl-DNA was included in the hybridisation buffer (Landegent, J.E., Jansen in de Wal, N., Dirks, R.W., Baas, F. and van der Ploeg, M. (1987) Use of whole cos- mid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridisation Hum. Genet. 77, 366-370) in order to avoid inserts containing repetitive sequences. Positive plaques were selected that, in addition, were not detected on a replica filter, which had been hybridised to a cocktail of frequently expressed, known, sequences. Probes were prepared by transcription of cDNA inserts after in vivo excision of pBluescript plasmids from the lambda ZAP phages (Stratagene protocol). Sequences expressed in marrow and bone forming cells were identified by in situ hybridisation (for a detailed protocol, see http://www.ki.se/cob): and partial sequencing followed by database searches identified so far unknown sequences among those. Fig. 1 shows results of in situ hybridisation experiments of sections through femur of a 5-day-old rat. Signal for RP59 RNA in a is observed in bone marrow (lower left) and in osteoblasts (arrows). Collagen type I RNA, in contrast, is a product typical for osteoblasts, but absent in most marrow cells. Control: Unrelated neomycin resistance sequence, showing non-specific background. Analysis of RP59 mRNA To assess the size and distribution of RP59 transcripts, Northern blots of poly-A enriched total rat RNA from different tissues were performed and hybridised to a RP59 probe (Fig. 2). Two bands of RP59 RNA are displayed, at 2.8kb and 2.6 kb. The difference between the RP59 bands is referred to two alternative poly-A addition sites, at NT 2147 and NT 2489 (SEQ ID NO:l); both are represented by inde- pendently isolated cDNA clones. This difference between the mRNA lengths depends thus on the non-translated part and a biological significance is so far not known. The faint band migrating at 4.5 kb is due to the presence of 28S RNA, which gives signal with all riboprobes. The size of the transcripts corresponds to the length of the derived cDNA sequence, if a poly-A track of 0.2 to 0.3 kb is assumed. Sequence of RP59 mRNA Several cDNA clones plus RACE fragments were aligned to determine the RP59 mRNA sequence. The complete cDNA sequence is displayed as SEQ ID NO: l : the putative translation start is at position 64 to 66, the stop codon at position 1510 to 1512. Thus, an open reading frame coding for a protein sequence of 482 amino acid residues was identified. (Table 1, SEQ ID NO:2). Starting with residue 144, a consensus sequence of 14 amino acids was repeated 14 times, arranged in 7 double-arrays of 28 amino acids each. The double-repeats were more similar to each other than the two repeats in each array. Two distantly related consensus sequences were derived for the first and second repeat (Table 2). This structure was followed by 42 (3 times 14) amino acid residues without obvious repetitive features, and then with 5 single repeats of 14 amino acid residues each. The consensus of those 5 repeat sequences had little similarity to both double-array sequences (Table 2). The 5' portion of the mRNA sequence, reaching from NT 1 to 23 and NT 49 to 537, was present in sequence databases as expressed sequence tag (EST) from rat embryo tissue. This entrance thus includes sequences coding for 14 amino acids that correspond to part of the first double repeat. However, coding capacity and partition in a repetitive sequence organization has not been indicated. Translation products A 20mer peptide with the sequence HSSKVPQDMAGPEALSLKMC (printed bold in Table 1) from the double-repeat region was synthetized by chemical means, coupled to keyhole limpet hemocyanine, and the complex was used to raise antibodies in rabbits (Neosystems, Strasbourg, France). This antiserum was used in order to determine the presence of the RP59 translation products. As a negative control, all reactions to protein extracts and histological sections were compared to reactions of preimmune serum from the same rabbit. A DNA fragment comprising nucleotide 493 to nucleotide 527 according to the sequence listing was amplified by the polymerase chain reaction, using oligonu- cleotides encompassing RP59-flanking restriction enzyme cleavage sites. The fragment was ligated into a bacterial expression vector and transformed into suitable bacteria (Qiagen-system). Bacteria were grown and the RP59 peptide was isolated under denaturing conditions by virtue of an affinity tag. Rabbits were immunized and antiserum was affinity purified by a affinity resin that contained coupled RP59 peptide. Western blots of femur extracts identified a unique band of 90 kd, both with the serum versus the chemically synthetized peptide and with the serum versus the artificially expressed protein fragment (see Table 1). Localization of RP59 RNA and protein in tissues Immunolabelling and in situ hybridization of histological sections revealed RP59 in the early and late embryo, in femoral bone marrow cells and in cells of spleen. Similar expression patterns were obtained with both experimental assays. At early embryonal stages, RP59 RNA is found in primitive mesenchymal cells, both in the primitive streak and in cells of the extraembryonal mesenchyme (Fig.5 A). At later stages, for example day 17 p.c, RP59 is present almost exclusively in the fetal liver, with the exception of some peripheral blood cells (Fig. 4). About 20% of all liver cells contain RP59 protein, dependent on the age and the relative proportion of non-labelled hepatocytes as well as in embryonal cells (Fig. 4, IHC). In adult spleen, RP59 protein is present in a portion of the red pulp, containing red blood cells, but absence from the white pulp that is composed of lymphatic cells. In femoral sections of 5 day-old rats, the antigen occurred at high concentration in marrow cells (arrowheads), and at low concentration in osteoblasts (thin arrows). No labelling of mature erythrocytes was observed (big arrows). It is concluded from these experiments that RP59 is a component of primitive mesenchymal cells and cells of the erythroid lineage, and that the protein is lost during erythrocyte differentiation. The presence in osteoblasts is interpreted to indicate recruitment from early erythroid stages.

Claims: CLAIMS 1. An isolated protein comprising essentially all of the amino acid sequence according to SEQ ID NO: 2, or a functional fragment or an analogue thereof, preferably originating from mesenchymal cells or primitive erythroid cells. 2. A fragment according to claim 1, which is characterised by the consensus sequence C-Q-E-(a)-T-V-P-Q-X- (b)- S - (c)- K - (a) wherein X denotes any amino acid; (a) denotes T or I; (b) denotes H or Y; (c) denotes P or S. 3. A fragment according to claim 1, which is characterised by the consensus sequence (d)-Q-D-M-A-(e)-P-E-A-L-S-P-K-(f) wherein (d) denotes C, P or H; (e) denotes G or E; and (f) denotes M or T. 4. A fragment according to claim 1, which is characterised by the consensus sequence L-S-H-K-T-X-(g)-E-X-X-(h)-P-(i)-X wherein X denotes any amino acid; (g) denotes Q or K; (h) denotes E or G; and (i) denotes E or K. 5. A fragment according to any one of claims 1-4, which is characterised by the sequence defined by amino acid nos. 262-281 as defined in SEQ ID NO:2. 6. A protein or polypeptide which comprises at least two of the fragments according to any one of claims 2-5. 7. A nucleic acid encoding a protein, polypeptide or fragment as defined in any one of claims 1-6. 8. A nucleic acid according to claim 7, which comprises essentially all of the nucleotide sequence according to SEQ ID NO: 1, or a variant of said sequence. 9. A nucleic acid, which is capable of specific hybridisation under stringent conditions with a sequence accordmg to claim 7 or 8. 10. An expression vector comprising the nucleic acid as defined in any one of claims 7-9. 11. An expression system comprising the nucleic acid as defined in any one of claims 7-9 and capable of mediating expression of a protein, polypeptide or fragment as defined in any one of claims 1-6. 12. An isolated or recombinant cell comprising a nucleic acid as defined in any one of claims 7-9. 13. A protein, polypeptide or fragment as defined in any one of claims 1-6 for use as a medicament, such as an antigen. 14. Use of a protein, polypeptide or fragment as defined in any one of claims 1-6 in the manufacture of a medicament. 15. Use according to claim 14, wherein said compound serves as affinity label for the isolation of cells, preferentially progenitor or stem cells. 16. A pharmaceutical preparation comprising a protein, polypeptide or fragment as defined in any one of claims 1-6 and a pharmaceutically acceptable carrier. 17. A method of treating and/or preventing connective tissue disorders, which comprises stem cells, isolated by virtue of a protein, polypeptide or fragment as defined in any one of claims 1-6. 18. A method of preparing an implant or a transcutanous device suitable for use in transplantation surgery, wherein cells as defined in claim 12 are used to improve the biocompatibility of said implant and/or transcutanous device, preferably by application of a coating layer. 19. An implant or a transcutanous device prepared by the method according to claim 18. 20. A compound capable of specifically recognizing a protein, polypeptide or fragment as defined in any one of claims 1-6 or a nucleic acid as defined in any one of claims 7-9, which compound is a peptide or an antibody, preferably an antibody. 21. Use of a nucleic acid as defined in any one of claims 7-9 or a compound as defined in claim 20 as a tool in diagnosis of disorders that concern, bone, bone marrow and/or blood. 22. A method of diagnosing bone, bone marrow or blood disorders, either genetical or aquired, which a nucleic acid as defined in any one of claims 7-9 or a compound as defined in claim 20, which method is applied in vivo or in vitro. 23. A detection system comprising a compound as defined in claim 21, wherein said compound is recognised in histological sections, Western blots, and/or test systems for the analysis of diagnostic samples.

Inventors: CHRISTERSSON CECILIA, LUNDMARK CARIN, WURTZ TILMANN, CHRISTERSSON, CECILIA, LUNDMARK, CARIN, WURTZ, TILMANN
Applicants: CHRISTERSSON CECILIA, CT FOER ORALBIOLOGI, LUNDMARK CARIN, WURTZ TILMANN, CENTRUM FOER ORALBIOLOGI, CHRISTERSSON, CECILIA, LUNDMARK, CARIN, WURTZ, TILMANN
Application year: 2000
Main classification: C07K14
IPC codes: A61K38, C07K14