CDNA Sequences of Human Bone Matrix Proteins

The present invention is related generally to providing cDNA sequences of proteins. More particularly, the present invention is related to providing cDNA sequences as well as sense and anti-sense RNA and DNA sequences and the deduced amino acid sequences of a plurality of human bone matrix proteins, specific antisera and various applications thereof.

Certain bone and connective tissue disease processes are detectable by monitoring the level of certain macromolecules in the serum. An example is serum osteocalcin, the levels of which reflect bone turnover. However, heretofore, only a limited number of such macromolecules were known to be related to bone and connective tissue diseases. The macromolecules cited herein comprise the majority (70% of the noncollagenous proteins in the human skeleton) that are truly of bone cell origin, that is the cDNA sequences were derived from screening of normal human bone cell cDNA libraries, and not from cDNA libraries of cells or tissues of other non-bone types, or of transformed cells. The levels in tissues or bodily fluids, such as serum, plasma, crevicular fluid, amniot fluid, urine, etc., of these molecules or their metabolites reflect normal bone metabolism. A shift in those levels from the normal values would be indicative of skeletal and/or connective tissue disease states.

It is, therefore, an object of the present invention to provide specific cDNA and deduced protein sequences which are diagnostic of such human diseases as osteoporosis, osteo/rheumatold arthritis, Paget's disease, atherosclerosis, periodontal disease and the like.

It is another object of the present invention to provide novel probes (sense and anti-sense RNA and DNA derived from the cDNA sequence), antibodies and bioassays for monitoring the level and distribution of certain human bone matrix proteins in the human body fluids or tissues.

Other objects and advantages will become evident from the following detailed description of the invention.

These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein:

FIGS. 1-6 show the cDNA and deduced amino acid sequences of human bone biglycan (proteoglycan I), human bone decorin (proteoglycan II), human bone osteopontin Ia and human bone osteopontin II, human bone sialoprotein and human bone osteonectin(ON), respectively.

The above and various other objects and advantages of the present invention are achieved by utilizing a part or the whole of the cDNA (sense and anti-sense sequences of RNA and DNA derived from the cDNA sequence) and protein sequences shown in FIGS. 1-6. It is noted that given the cDNA and protein sequences shown in FIGS. 1-6, one of ordinary skill in the art can easily prepare or derive specific sequences (sense and anti-sense RNA or DNA) of the cDNA or polypeptides for diagnostic tests or bioassays by standard techniques well known in the art and can be found described in such publication as Maniatis et al (A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982); Davis et al (Basic Methods in Molecular Biology. Elsevier, N.Y., 1986); Ausubel et al (Current Protocols in Molecular Biology. Greene Pub. Assoc. and Wiley Interscience, N.Y., 1987); Koziolkiewicz et al (Chem. Sci. 26: 251-260, 1986); van Regenmortel et al (Laboratory Techniques in Biochemistry and Molecular Biology. Vol 19. Synthetic Polypeptides as Antigens. Amsterdam, Elsevier, 1988); Robey and Fields (Anal. Biochem. 177: 373-377, 1989).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. It is noted that the methods, materials and examples are only illustrative and not limiting.

The term "substantially pure" as used herein means that the product is as pure as can be obtained by employing conventional isolation and purification techniques known in the art.

Construction and Screening of a Bone Cell Culture Derived cDNA Library

RNA from primary cultures of adult human bone cells (Gehron Robey et al. Calcif. Tissue Int. 37: 453-460, 1985) was extracted using a guanidine HCl procedure (Adams et al. Proc. Natl. Acad. Sci. U.S.A. 74: 3399-3403, 1977), and poly(A).sup.+ mRNA was isolated by affinity chromatography on oligo(dT)-cellulose (Pharmacia LKB Biotechnology Inc. ). Approximately 20 .mu.g of poly(A).sup.+ was used to construct a gt11 ZAP library (custom library section of Stratagene Cloning Systems). The amplified cDNA expression library was first screened in Escherichia coli BB4 cells as described by Young and Davis (Proc. Natl. Acad. Sci. U.S.A. 80: 1194-1198, 1983) using for example a polyclonal antiserum from a rabbit injected with both human bone PG I and PG II (rabbit LF-5) and a peroxidase-conjugated second antibody made in goat (Kirkegaard and Perry Laboratories). Positive plaques were identified by reaction with H.sub.2 O.sub.2 and 4-chloro-1-naphthol. Positive clones were rescreened to purity with the same antiserum and then screened again in a second round with antisera made against synthetic peptides corresponding to residues 11-24 of the secreted form of human bone PG I (Fisher et al. J. Biol. Chem. 262: 9702-9708, 1987) or 5-17 of human fibroblast PG II (Krusius and Rouslahti. Proc. Natl. Acad. Sci. U.S.A. 83: 7683-7687, 1986). The synthetic peptides were made on an Applied Biosystems Model 430A peptide synthesizer using t-butoxycarbonyl-protected amino acids and standard reaction conditions suggested by the manufacturer. The peptides were deprotected using anhydrous HF and conjugated either to bovine serum albumin for PG I (LF-15) or to keyhole limpet hemocyanin for PG II (LF-30), respectively, as described (Lindner and Robey. Int. J. Peptide Protein Res. 30: 794-800, 1987). Clones containing cDNA for PG I were found to be missing the start codon of the open reading frame, so the library was rescreened using a 200-bp 5'-EcoRI-BglII fragment labeled with .sup.32 P (Amersham nick translation kit and 3000 Ci/mmol of [.sup.32 P]O.sub.4 .alpha.-CTP, Du Pont-New England Nuclear ). Prehybridization, high stringency hybridization, washing, and detection on Kodak XOmat AR film were as described (Young et al. Nucleic Acids Res. 12: 4207-4228, 1984). Bone sialoprotein (BSP) and osteopontin (OP) I and II were screened using 32P-labeled rat BSP and OPI, respectively, under moderate stringency conditions (60 C.).

DNA Sequence of cDNAs

Purified insert(PGI, PGII, BSP, OPI,OPII and ON) was isolated from plasmid DNA according to the instructions provided by Stratagene Cloning Systems. Briefly, the purified phage vectors containing the cDNAs were used to co-infect a BB4 E. coli strain with R408 helper phage (Stratagene Clong Systems ). The pBluescript plasmid DNA (packaged in M13 or f1 phage particles at this stage) was rescued by infecting and plating fresh bacteria on ampicillin-containing plates. (Phage in the helper phage preparation were destroyed by heating to 70.degree. C. for 20 min. ) Colonies (containing the pBluescript plasmid and the cDNA insert) were plucked and grown in a larger scale preparation. As a specific example, PGI cDNA inserts were liberated either with a combination of BamHI and KpnI or XbaI and NaeI (the latter removing only a .about.1.O-kbp 5' piece). These large fragments were directionally subcloned in M13mp18 or -19 (Messing et al. Nucleic Acids Res. 9: 309-321, 1981 ) previously restricted with the above enzymes (using SmaI for the NaeI site). Smaller restriction fragments were also made from the XbaI/NaeI fragment using BglII and RSaI and subcloned into appropriately cut M13 vectors. After transformation into JM101 cells, single-stranded DNA from recombinant phage was annealed to either the 17-bp universal primer or the appropriate synthetic oligonucleotide of an internal site (Messing et al. supra) and sequenced using the dideoxy chain termination method (Sanger et al. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467, 1977). The SEQUENASE kit (United States Biochemicals ) using both GTP and ITP nucleotide mixes and .alpha.-[.sup.35 S]ATP (1110 Ci/mmol, Du Pont-New England Nuclear) were used for the sequencing reactions of PGI and all other clones. The nucleotide sequences were determined by electrophoresis on 6% and 8% polyacrylamide urea gels followed by exposure on X-AR film (Kodak).

Northern Analysis

3.5-.mu.g portions of total RNA from cells in culture (human bone, skin, periodontal ligament, gingiva, bovine bone, chicken embryo flbroblasts, rat osteosarcoma (ROS 17/2), or tissues (bovine: skin, articular cartilage, and cornea; and rat swarm chondrosarcoma) were electrophoresed in 1.2% formaldehyde-agarose gels and transferred to nitrocellulose as described (Maniatis et al. Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982). An agarose gel-purified .XbaI-NaeI fragment of PG I (clone P6, included >90% of the coding region of the cDNA but no noncoding regions and corresponded to nucleotides .about.180-1130) or PG II (clone P2, entire BamHI/KpnI 1.6-kbp insert) were labeled by nick translation (see above) and hybridized to the RNA bound to the nitrocellulose. Hybridization was carried out at 37.degree. C. in 40% formaldehyde for 16 h, washed, and exposed to x-ray film for 3-6 h as described previously (Shimokawa et al. J. Biol. Chem. 262: 4042-4047, 1987). A blot was probed for PG I mRNA, autoradiographed, stripped of PG I probe by boiling for 5 min in 2.times.SSC, and 0.1% SDS followed by an ice-cold wash in 2.times.SSC, and reprobed with the PG II cDNA probe.

Polyacrylamide Gel Electrophoresis and Electroblotting

Polyacrylamide gradient (4-20%) SDS slab gels (160.times.140.times.1.5 mm) topped with 3% stacking gel were prepared and electrophoresed as described (Fisher et al. J. Biol. Chem. 258: 6588-6594, 1983). Electrotransfer of core proteins (in 150 .mu.g dry weight of adolescent monkey bone mineral compartment extract; 200 .mu.g each of 4M guanidine HCl-extracted monkey skin, tendon, cartilage, cornea, or muscle (crude extracts); or 10 .mu.g of purified human bone PG I (Fisher et al. J. Biol. Chem. 262: 9702-9708, 1987) each digested for 1 h at 37.degree. C. with 10 milliunits of chondroitinase ABC (Miles) (Fisher et al, supra) from the SDS gels and onto nitrocellulose was according to the method of Towbin et al (Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354, 1979). Indirect immunodetection using antisera and conjugated second antibodies was as above.

Probes derived from the cDNA sequence (either sense or anti-sense RNA or DNA, partial or full length), and polyclonal or monoclonal antibodies having specific binding affinity to the amino acid sequences (either synthetic peptide, fusion proteins or full length naturally occuring proteins) of the human bone matrix proteins of the present invention are produced by standard techniques well known in the art. These inventions are used in procedures as noted below.

Immunoassays (RIA and ELISA) with Antibodies Raised Against Either Intact Protein or Synthetic Peptides

The invention (synthetic peptide or substantially pure protein, and antibodies raised against either or both) is used in a radioimmunoassay (RIA) to measure levels of the bone matrix proteins or their biosynthetic or degradative products in bodily fluids or tissue extracts (Risteli, et al. In Kuhn, K., Krieg. T. (eds) Connective Tissue: Biological and Clinical Aspects. Munich, Karger. 1986, pp.216-245). First the synthetic peptide or substantially pure protein is used to establish a standard curve by dissolving an accurately measured amount in an appropriate buffer and diluted to yield a series with varying concentrations. The assay tubes (total volume of 200 microliters) for establishing the standard curve contains 100 microliters of the prepared standard (each tube containing a different amount of polypeptide or protein), 50 microliters of the antibody diluted to an appropriate concentration and 50 microliters of iodinated, or otherwise labeled synthetic peptide or substantially pure protein (.about.10,000 cpm). The assay tubes for samples to be analyzed contain 50 microliters of buffer, 50 microliters of the analyte, 50 microliters of the diluted antibody and 50 microliters of the labeled peptide or protein. All tubes are incubated overnight at 4.degree. C. Each tube then receives 50 microliters of S. aureus bearing protein A (or any other solid support that binds to antibody) suspended in buffer. The complex is allowed to form during a 2 hour period of tube rocking at 4.degree. C. The immune complexes are then precipitated by centrifugation. The supernate is aspirated and the radioactivity of the pellet determined by scintillation counting. The standard curve is generated by quantitating the amount of radioactivity bound in the immune complex in the presence of increasing amounts of unlabeled peptide or protein. As the amount of unlabeled polypeptide or protein increases, the labeled polypeptide or protein bound decreases since the concentration of antibody is constant. Plotting of the amount of radioactivity vs. the amount of unlabeled polypeptide or protein results in a curve with a linear region. The level of polypeptide or protein in the analytes is determined from the standard curve.

The invention (either polypeptides or pure protein, and antibodies raised against either or both) is also used in direct or competition ELISAs (Enzyme-linked immunosorbent assay) (Rennard et al. Anal. Biochem. 104: 205-214, 1980). In a typical direct ELISA, a standard curve is established by binding a known concentration of peptide or pure protein (200 microliters) to the bottom of flat bottomed polyvinyl 96 well plates and serially diluting with Vollers buffer. The analyte is also bound at several different dilutions. The plate is incubated at 4.degree. C. overnight, and then washed three times with phosphated buffered saline containing 0.054 a detergent such as TWEEN. The antibody raised against either the peptide or pure protein is used in either a direct (the antibody itself is tagged with a reporter molecule, which is usually an enzyme such as horseradish peroxidase, etc. ) or indirect procedure, and is diluted to an appropriate concentration with PBS+Tween, 200 microliters is added to each well, and the plate is incubated at room temperature (.about.22.degree.-24.degree. C.) for one hour. The plate is washed three times with PBS+Tween. If the antibody itself is tagged, the appropriate substrate (which will form a chromophore after it is acted on by the enzyme) is added, but if an indirect detection procedure is used, a second antibody (raised against the Ig fraction of the animal species used to elicit the first antibody, and tagged with a reporter) is diluted to an appropriate concentration and 200 microliters are added to each well. The plate is incubated for 1 hr at room temperature, and washed three times with PBS+TWEEN. The appropriate substrate for the reporter is then added. After the addition of substrate, the plate is incubated for 1-6 hrs, and terminated by addition of a color development inhibitor such that the resultant optical density values are within the linear range of the detection system. The amount of chromophore generated is determined by spectrophotometry, and the standard curve is determined by plotting the optical density vs. the amount of polypeptide or protein. The values for the analyte are determined from the standard curve.

In a typical competition ELISA, the antibody against a polypeptide or pure protein is diluted to an appropriate concentration and 100 microliter aliquots are first incubated in a round bottomed, non-adsorptive 96 well plate with different amounts of polypeptide or pure protein (to generate the standard curve), and the analyte at various dilutions (final volume). After incubation at 4.degree. C. overnight, the aliquots are transferred to a 96 well flat-bottomed plate that has been previously treated by adsorbing a constant amount of polypeptide or antigen to the bottom of each well. The plates are then incubated, washed, etc. as described in the previous paragraph. The level of polypeptide or protein in an analyte is estimated from its ability to inhibit the binding of this antibody (which is either directly tagged with a reporter, or used in conjunction with a second antibody that is tagged) in comparison to the standard curve (which is generated by measuring the ability of known amounts of polypeptide or pure protein to competitively inhibit binding).

Immunohistochemistry with Antibodies Raised Against Intact Antibodies or Synthetic Peptides

In this assay, antibodies (either monoclonal or polyclonal ) raised against the invention (any synthetic polypeptide, or substantially pure protein preparation) are used to detect the localization (in a semi-quantitative fashion) within tissue sections (either frozen or fixed with an appropriate fixative) or cell culture preparations (either frozen or fixed). Tissue sections are processed and embedded in paraffin (for light microscopy) or Epon (or equivalent) (for electron microscopy) and sections of appropriate thickness are prepared. The specimen (either sections or cells) is prepared for immunohistochemistry by equilibrating in the physiological buffer and incubating with the antibody raised against the polypeptide or pure protein, diluted to an appropriate concentration. In cases of direct immunodetection, the specific antibody itself is covalently coupled to a reporter molecule (e.g., horseradish peroxidase, alkaline phosphatase, biotin or any other reporter). In cases of indirect immunodetection, a second antibody, raised against immunoglobulins derived from the animal species in which the specific antibody was elicited is covalently coupled to the reporter, and incubated with the specimen. After extensive washing the appropriate substrate for the reporter is added (e.g., diaminobenzidine as the substrate for horseradish peroxidase) and incubated for an appropriate amount of time relative to a positive control. The reaction is terminated, and the specimen is examined by light or electron microscopy and compared to a normal control specimen for evaluation (Timpl and Risteli. In Furthmayr, H. (ed) Immunochemistry of the Extracellular Matrix, Vol. 1 Boca Raton, CRC Press, 1982, pp.199-235; Bianco et al. In: Proceedings of the First Joint Meeting of the International Conference on Calcium Regulating Hormones and the American Society for Bone and Mineral Research, Elsevier Sci. Pub. Co., In Press ).

Use in Competition or Inhibition Assays, and Blocking of Biological Function

The invention (polypeptides or protein) is used in various competition assays as noted above for RIA and ELISA assays. Also they are used in inhibition of antibody localization in immunohistochemistry (a method of proof of specific antibody reaction) by first incubating the primary antibody with an excess amount of polypeptide or protein. The antibody is then incubated with the tissue sections or cells as described above. Blocking of the immunolocalization is indicative of specific reaction. In addition, polypeptides or protein are used to determine the presence or absence of an antibody in a test solution (e.g., patient's bodily fluids as In the case of pathological conditions such as autoimmunity, or in hybridoma growth medium or ascites fluid in the preparation of monoclonal antibodies).

The invention (polypeptides or protein) is used to either induce biological function, or to block it. In the case of cell attachment, the invention is adsorbed or crosslinked onto an inert support and used either in vivo or in vitro. The invention can be used to block cell attachment by incubating cells in suspension (or with a systemic delivery) which thereby inhibits reaction of cells with the substratum (e.g., Pierschbacher and Rouslahti, Nature #09: 30-34, 1984).

The Invention (anti-sense sequences derived from the cDNA or any derivative thereof (e.g., phosphorothioate, methylphosphonate, or in combination with targeting molecules) are used to block biological function by inhibition of protein synthesis. The anti-sense sequences are used in vivo by topical application or may be used systemically when covalently attached to a targeting agent that provides cell and tissue specific localization. vitro, they are used by addition to tissue culture medium, and are actively taken up by the cells, where they inhibit protein synthesis, thereby blocking biological function of the invention (Marcus-Sekura. Anal. Biochem. 172:289-295, 1988).

In situ Hybridization

The invention (anti-sense sequence of the cDNA) is used in in situ hybridization for the localization of mRNA within tissue sections and cell preparations. Typically, sections of tissues or cells (permeabilized by treatment either with detergent or Saponin) are fixed, embedded and sectioned as described above. Deparaffinized sections are pretreated under acid conditions prior to digestion with proteinase K, followed by acetylation with acetic anhydride in triethanolamine. The tagged (usually radiolabeled) anti-sense sequence (partial or full length) is added to the section in hybridization buffer and incubated overnight at 50.degree. C., washed and treated with RNase to remove non-specifically bound probe, and then washed under increasingly stringent conditions. If the probe is radiolabeled, sections are subjected to autoradiography using Kodak NTB-2 emulsion. If the probe is tagged with another reporter, an appropriate detection system is used (Metsaranta et al. Calcif. Tiss. Int. 45: 146-152, 1989).

Northern Blot Analysis

The invention (antisense sequence derived from the cDNA) is used in Northern blot analysis to determine in a semi-quantitative fashion the presence of mRNA in extracts of tissues and cells. Total RNA is extracted and electrophoresed on formaldehyde agarose gels, and then transferred to nitrocellulose. The "Northern" blot is washed in prehybridization solution, and subsequently hybridization is carried out using a radiolabeled (or otherwise tagged) cDNA probe at 41.degree. C. Then hybridization is visualized by standard autoradiography or any suitable detection system well known to one of ordinary skill in the art.

A diagnostic kit in accordance with the present invention which can be used in conjunction with RIA, ELISA, immunohistochemistry and the like, comprises separate containers containing:

a. An appropriate buffer for standards and samples.

b. A sample of the invention (polypeptide or protein) of known concentration.

c. A sample of antibody (either monoclonal or polyclonal) raised against the invention (polypeptide or protein) to be used at an appropriate dilution.

d. A buffer for the dilution of the antibody.

e. A sample of radiolabeled or otherwise tagged polypeptide or protein (the invention).

f. A solid bead or resion having a substrate covalently attached to it that will bind the specific antibody.

g. A sample of non-immune serum as a negative control, and instructional material for performing the test.

A bioassay in accordance with the present invention such as RIA, ELISA, immunohistochemistry and the like for detecting the level of certain human bond matrix proteins, comprises the steps of:

a. Preparing standards--The lyophilized polypeptide or protein of the present invention is resuspended in a buffer to yield a concentrated stock solution. The stock solution is diluted with buffer to yield standard solutions within a given range of protein or polypeptide concentration.

b. Preparing tubes for establishing the standard curve--In separate tubes, 100 microliters of each of the standard dilution samples is added along with 50 microliters of the antibody diluted in antibody buffer, and 50 microliters of radiolabeled polypeptide or pure protein.

c. Preparing tubes of samples to be analyzed--To separate tubes, add 50 microliters of buffer, 50 microliters of sample to be analyzed (at several different dilutions), 50 microliters of antibody diluted with antibody buffer, 50 microliters of radiolabeled polypeptide or pure protein.

d. Incubate at 4.degree. C. overnight.

e. Add 50 microliters of solid support to capture the antibody-antigen complex. Incubate at 4.degree. C. with constant agitation.

f. Centrifuge at 10,000.times. g for 5 min. Draw off supernatant, wash pellet with buffer and recentrifuge. Draw off supernatant.

g. Quantitate radioactivity of pellet.

h. Construct a standard curve by plotting the radioactivity in the standard tubes (y axis ) vs. amounts (x axis ).

i. Determine concentration of polypeptide or protein in the sample by placing the radioactivity on the curve at the appropriate y value and reading the corresponding x value (amount).

A probe in accordance with the present invention for detecting the presence of mRNA of certain bone matrix protein, comprises employing the anti-sense sequence derived from the cDNA sequence of said bone matrix protein as a probe.

A sequence for inhibiting protein synthesis comprises employing the anti-sense sequence of the cDNA (either partial or full length) and any chemically modified derivative of the cDNA sequences of the bone matrix protein of the present invention.

An antibody having specific binding affinity for the polypeptides of the present invention is easily made by conventional monoclonal or polyclonal techniques well known to one of ordinary skill in the art.

A deposit of the clones has been made at the ATCC, Rockville, Md., on Sep. 28, 1989, under the accession numbers as follows:

The deposit shall be viably maintained, replacing if it becomes non-viable during the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and upon issuance of the patent made available to the public without restriction in accordance with the provisions of the law. The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.