# Patent

## Title
Bone metabolism related protein and gene thereof

## Abstract

The present invention is directed to a novel method of detecting a function or activity of a polypeptide which is related to bone metabolism, in particular, differentiation (maturation) of osteoblast or morphological change (retraction), specifically relating to a polypeptide which comprises an amino acid sequence shown by SEQ ID NO: 2 or SEQ ID NO: 4, an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or SEQ ID NO: 4, or a polypeptide encoded by a nucleic acid which is capable of hybridizing under stringent condition with a nucleic acid comprising a nucleotide sequence shown by SEQ ID NO: 1 or SEQ ID NO: 3, or a complement sequence thereof.

## Background
This application claims priority on provisional Application No. 60/292,318 filed on May 22, 2001, the entire contents of which are hereby incorporated by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel protein and a gene thereof which is involved in bone metabolism. Further, the present invention relates to a novel protein and the gene thereof having a function or an activity of (i) promoting differentiation of osteoblast, (ii) inducing morphological change of a cell or (iii) an esterase activity, etc.
2. Prior Art
Normal bone metabolism depends on a balance between bone formation and born resorption. It has been known that bone formation is mainly led by osteoblast that is differentiated from a messenchymal stem cell and bone resorption is led by osteoclast that is differentiated from a hematopoietic stem cell.
It is thought that osteoporosis is caused when this balance is shifted to the bone resorption side. Osteoporosis is classified into two types. One is called postmenoposal osteoporosis in which remarkable decrease in bone mass is observed. In this type, bone metabolism is at a high turnover rate, and bone formation and bone resorption are both active. However, the balance is shifted toward the bone resorption side, thereby causing osteoporosis. The other type is called a senile osteoporosis in which a decrease in bone mass is caused gradually. In this type, a cause is thought to be a dysfunction of the osteoblast, which leads to a declined balance toward the bone resorption side.
There have been many points that are left unclear about mechanisms of the bone formation or of differentiation of osteoblasts, for example, as a factor that is involved in differentiation of osteoblasts, only a few have been known, such as bone morphogenetic protein (BMP) (Maiti, et al., Indian J. Exp. Biol., vol. 36, pp. 237 to 244, 1998), a transcription factor Cbfal (Komori, et al., Cell, vol. 89, pp. 755 to 764, 1997), etc.
On bone remodeling, there have been known facts as follows. That is, when a concentration of calcium ion in blood is lowered, secretion of parathyroid hormone (PTH) from accessory thyroid gland is increased, and PTH directly acts on bone, causing bone resorption and calcium ion release. In this process, it is known that PTH acts on osteoblasts and induces morphological change of the cells. There is a hypothesis advocating that a part of a bone surface covered by osteoblasts is exposed due to such morphological change of the osteoblasts, thereby providing a space for osteoclasts to adhere to (Rodan et al., Calcit. Tissue Int., vol. 33, pp. 349 to 351, 1981; “Principles of Bone Biology” (J. P. Bilezikian, L. G. Raisz, G. A. Rodan, eds.), 1996, Academic Press Inc., USA.). At an initial stage of bone resorption of bone remodeling, adhesion of osteoclasts to the bone surface is of importance, and, morphological change of osteoblasts is also as important. Despite of this, there has not been known much about a detailed mechanism of morphological change of osteoblasts during bone remodeling.
It has been earnestly desired that these mechanisms are solved for research and development of a therapeutic treatment method and a therapeutic agent for diseases related to bone metabolism, such as osteoporosis, etc.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a novel protein and a novel gene which relate to bone metabolism. Another object of the present invention is to provide a means for detecting these functions or expressions. Still further object of the present invention other than the above will be clarified by the following descriptions.
The present inventors have found a novel protein and its gene which is specifically expressed when osteoblast-like cells (mouse MC3T3-E1) differentiate (mature) to osteoblasts having an active bone morphogenetic potential. Moreover, they have found that differentiation (maturation) to osteoblasts is promoted by overexpression of this gene in the osteoblast-like cells, and that morphological change of the cells occurs thereby, etc., and then, they have accomplished the present invention.
That is, the present invention relates to a polypeptide which comprises a polypeptide selected from the following (A), (B) and (C), and has a function or an activity selected from the following (i), (ii) and (iii):

    (A) a polypeptide comprising an amino acid sequence shown by SEQ ID NO: 2 or SEQ ID NO: 4,
    (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or SEQ ID NO: 4,
    (C) a polypeptide encoded by a nucleic acid which are capable of hybridizing under stringent condition with a nucleic acid comprising a nucleotide sequence shown by SEQ ID NO: 1 or SEQ ID NO: 3 or a complement sequence thereof,
    (i) promoting differentiation (maturation) of osteoblast,
    (ii) inducing morphological change (particularly retraction of osteoblast) of a cell, and
    (iii) an esterase activity (particularly a glycerophosphodiester phosphodiesterase activity or the like).


Also, the present invention relates to a nucleic acid which encodes the above-mentioned polypeptide.
Moreover, the present invention relates to a recombinant vector and a host cell containing the same. Furthermore, the present invention relates to a method of detecting a function or an activity of the polypeptide or the nucleic acids by using the above-mentioned polypeptide or the nucleic acids. Additionally, the present invention relates to a method for screening or identifying a compound that shows an effect of modulating a function or an activity (or an expression) of the polypeptides (or the nucleic acids), using the same.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an illustration showing a predicted structure of OBDPF protein as a transmembrane protein.
FIG. 2 is a restriction enzyme map of a mouse OBDPF genomic DNA, and a drawing showing positions of its exons. The HindIII sequence is SEQ ID NO: 17.
FIG. 3 is a drawing showing an expression of OBDPF gene (a result of Northern blotting) in mouse MC3T3-E1 cells induced to promote differentiation (after 0, 4, 7, 11 and 15 days of culture following addition of ascorbic acid and β-glycerol phosphate).
FIG. 4 is a drawing showing an expression of OBDPF gene (a result of Northern blotting) in various tissues in mouse.
FIGS. 5A and 5B are graphs showing results of analysis of osteoblast differentiation marker in mouse MC3T3-E1 cells in which OBDPF gene is overexpressed ((A) alkaliphosphatase activity 0 to 14 days after culture, (B) amount of calcium deposited after 14 days of culture, respectively).
FIG. 6 is a drawing showing homologies in amino acid sequence of OBDPF and known enzymes, SEQ ID NOS:13–16.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
Osteoblasts are cells that are responsible for bone formation, and generally exist on a forming surface of a bone that undergoes growth.
The protein (Osteoblast Differentiation Promoting Factor: OBDPF) and the gene thereof (Osteoblast Differentiation Promoting Factor gene: OBDPF gene) found by the present inventors are expressed specifically in a stage where osteoblasts undergo differentiation (maturation). That is, there is no expression or a low level of expression of the OBDPF or a gene thereof observed when osteoblasts are in undifferentiated (immature) state (a state where they do not have an active bone morphogenetic potential), but there is a higher level of expression observed when osteoblasts are in differentiation (matured) state (a state where they acquire an active bone morphogenetic potential) than in undifferentiated (immature) state.
The OBDPF protein and the gene thereof have a function to promote differentiation (maturation) of osteoblasts. That is, expression thereof promotes transferring those cells from an undifferentiated (immature) state to a differentiated (matured) state.
Further, OBDPF protein and the gene thereof have a function to induce morphological change of the cell (specifically, retraction of osteoblasts). That is, intracellular expression thereof changes a shape of the cell more spherical.
In addition, OBDPF protein has an enzyme activity (an esterase activity, in more detail, glycerophosphodiester phosphodiesterase activity).
SEQ ID NO: 1 of the sequence listing mentioned below is a nucleotide sequence of a cDNA of a mouse OBDPF gene isolated by the present inventors, and SEQ ID NO: 2 is an amino acid sequence of an OBDPF protein encoded in the coding region thereof, respectively. SEQ ID NO: 3 is a nucleotide sequence of a cDNA of a human OBDPF gene, and SEQ ID NO: 4 is an amino acid sequence of an OBDPF protein encoded in the coding region thereof, respectively. When comparison is made between amino acid sequences (539 amino acid residues) of human and mouse OBDPF proteins, there is about 87% of homology.
SEQ ID NO: 5 to 12 show a DNA sequence containing exon-intron boundary region in a genomic DNA of the mouse OBDPF.
As a protein or a polypeptide of the present invention, there may be mentioned those with an amino acid sequence shown by SEQ ID NO: 2 or NO: 4, and those with an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or NO: 4. Deletion, substitution or addition of the amino acids may be allowed to be of such a degree that a function or an activity thereof is not lost (for example, a function to promote differentiation of osteoblasts, a function to induce morphological change of a cell and or an esterase activity), and it is generally 1 to about 110 amino acids, preferably 1 to about 55 amino acids, more preferably 1 to about 30 amino acids.
Such a protein or a polypeptide has a homology in an amino acid level of generally about 80% or more, preferably about 90% or more, more preferably about 95% or more to the amino acid sequence shown by SEQ ID NO: 2 or NO: 4. Such a protein or a polypeptide includes an artificially modified mutant protein, proteins originated from other living organisms and the like, in addition to a mutant protein discovered in nature.
These proteins or polypeptides are exemplified by a polypeptide comprising an amino acid sequence having one or more conservative amino acid substitutions in comparison with the amino acid sequences shown by SEQ ID NO: 2 or NO: 4, and this includes conservative substitution variants and naturally occurring allelic variants of the polypeptide having an amino acid sequence shown by SEQ ID NO: 2 or NO: 4.
As the gene or the nucleic acid of the present invention, there may be mentioned a nucleic acid having a nucleotide sequence shown by SEQ ID NO: 1 or NO: 3. Further, there may be mentioned a nucleic acid which is capable of hybridizing under stringent condition with a nucleic acid having a nucleotide sequence shown by SEQ ID NO: 1 or NO: 3. Such a nucleic acid that is capable of hybridizing may be any as long as functions (for example, a function to promote differentiation of osteoblasts, a function to induce morphological change of a cell, a function to express esterase activity, and the like) thereof are not lost. Such a nucleic acid has a homology of generally about 70% or more, preferably about 80% or more, more preferably about 90% or more to the nucleotide sequence shown by SEQ ID NO: 1 or NO: 3. Such a gene or a nucleic acid includes a mutant protein discovered in nature, an artificially modified mutant gene, homologous genes originated from other living organisms, etc., and nucleic acids derived therefrom.
As the protein or the polypeptide in the present invention, there may be mentioned those which are recombinant type or those which are isolated.
As the nucleic acid in the present invention, there may be mentioned a DNA molecule and an RNA molecule, and included are those which are recombinant type or those which are isolated. Further, these nucleic acids include a single stranded or double stranded nucleic acid. More specifically, for example, the nucleic acid comprising a nucleotide sequence shown by SEQ ID NO: 1 (or NO: 3) includes a single stranded DNA having said nucleotide sequence, a double stranded DNA comprising the single stranded DNA having said nucleotide sequence and the complement thereof, RNA molecules corresponding thereto, etc.
In the present invention, hybridization under stringent condition can be generally carried out by conducting hybridization for 16 hours in 6×SSC or a hybridization solution having an equivalent salt concentration at a temperature of 50 to 60° C., followed by preliminary washing with 6×SSC or a solution having an equivalent salt concentration as necessary, and subsequently washing with 1×SSC or a solution having an equivalent salt concentration. Further, under a condition with a higher stringency (high stringent condition), washing is carried out with 0.1×SSC or a solution with an equivalent salt concentration to conduct hybridization.
The gene or the nucleic acid of the present invention can be isolated and obtained by screening, using osteoblasts of mammals as a genetic source. As mammals, there maybe mentioned a non-human animals such as dog, cow, horse, goat, sheep, ape, pig, rabbit, rat and mouse, as well as human.
As osteoblasts, for example, osteoblasts isolated from bone (calvaria, etc.) of mammal as mentioned above can be used. Also, a cell line of osteoblast-like cells such as mouse osteoblast-like cell line MC3T3-E1 (Sudo, H. et al., Journal of Cell Biology, vol. 96, pp. 191–198, 1983; RIKEN RCB No.1126) can be used as osteoblasts. Or else, it is possible to use a cell which is able to be differentiated into osteoblast, example of which includes human osteosarcoma Saos-2 (RIKEN RCB No.0428) and the like.
The gene or the nucleic acid of the present invention can be isolated using a technique of selectively screening differentially expressed genes, such as a differential display method, a subtraction method, a differential hybridization method, etc., together with a differentiation model of osteoblasts. As the differentiation model of osteoblasts, in vitro culture system of osteoblasts as mentioned below can be employed.
For example, the osteoblast-like cells (mouse osteoblast-like cell line MC3T3-E1, etc.) are cultured in the presence of stimulating agent such as ascorbic acid and β-glycerol phosphate, etc., in order to induce differentiation thereby obtaining differentiated (matured) osteoblasts. Using mRNAs prepared from them, differentially expressed genes, whose expression amount differs between cells before differentiation (maturation) and those after differentiation (maturation), are screened by a method such as differential display method (Science, vol. 257, pp. 967–971, 1992 and Cancer Research, vol. 52, pp. 6966–6968, 1992) to obtain cDNA.
Using the obtained cDNA as a probe, cDNA library is screened by suitably combining a colony hybridization method, a plaque hybridization method and others to obtain entire length of cDNA. Further, by screening a genomic DNA library, genomic DNA (gene) can be isolated. In addition, homologous genes of other species of living organisms can be isolated by screening DNA libraries of other species of mammals. As such mammals, there may be mentioned a non-human animals such as dog, cow, horse, goat, sheep, ape, pig, rabbit, rat and mouse, as well as human.
Also, the gene or the nucleic acid of the present invention can be easily obtained using sequence information disclosed in the present specification (SEQ ID NO: 1 to 12 in the sequence listing shown below). For example, based on the information of the disclosed nucleotide sequence, a primer and a probe are designed and a DNA library is screened by suitably combining a PCR (polymerase chain reaction) method, a colony hybridization method, a plaque hybridization method and the like, thereby obtaining the gene or the nucleic acid of the present invention. DNA library such as cDNA library, genomic DNA library, etc., can be prepared by a method described in, for example, “Molecular cloning” (written by Sambrook, J., Fritsch, E. F. and Maniatis, T., published by Cold Spring Harbor Laboratory Press, 1989). In addition, if there is a commercially available library, it may be also used.
By determining nucleotide sequence of the obtained cDNA, the coding region encoding a protein that is a gene product can be determined, and an amino acid sequence of the protein can be obtained.
Moreover, using cDNA as a probe, northern blotting can be conducted with respect to mRNAs of undifferentiated cells and differentiated cells to confirm that the gene or the nucleic acid of the present invention is specifically expressed in a differentiation stage of osteoblasts.
A function of the protein or the polypeptide (or the gene or the nucleic acid) of the present invention can be detected as mentioned below.
i) A function of Promoting Differentiation (Maturation) of Osteoblast
For example, an expression vector to express the protein or the polypeptide (or the gene or the nucleic acid) of the present invention is introduced into undifferentiated (immatured) osteoblast and the gene is overexpressed. The overexpressed cells are cultured and a marker indicating differentiation of osteoblast is detected and measured to analyze a state of differentiation (maturation).
Osteoblasts with an active bone morphogenetic potential, namely, differentiated (matured) osteoblasts are recognized by an intracellular bone matrix deposit (deposited calcium), and they undergo calcification. Also, there exist granules exhibiting an alkaliphosphatase activity in the cytoplasm. When an active bone formation stops, it is known that those granules in osteoblasts disappear and alkaliphosphatase activity is suddenly lowered.
Therefore, as a marker for differentiation, an amount of calcification or an alkali phosphatase activity is properly used. In addition, osteocalcin activity or an amount of expression of osteopontin can be also used as a differentiation marker.
As osteoblasts, osteoblasts separated from calvaria of mammal or osteoblast-like cells (mouse osteoblast-like cell line MC3T3-E1, etc.) may be used, among which osteoblast-like cells (mouse osteoblast-like cell line MC3T3-E1, etc.) are especially preferably used.
ii) A Function of Inducing Morphological Change of Cells
For example, an expression vector designed for expressing the protein or the polypeptide (or the gene or the nucleic acid) of the present invention is introduced into cells (osteoblasts and the like) and expressed. These cells are cultured and observed with respect to morphological change (retraction). If the shape of the cell becomes spherical (retract), it is confirmed that it has a function of inducing morphological change.
iii) An Esterase Activity
For example, an enzyme activity is measured using cells in which the protein or the polypeptide of the present invention is expressed. Or else, the protein or the polypeptide may be used for the measurement of the enzyme activity after being isolated.
Since the fifth loop of the protein that is an extracellular region is responsible for an enzyme activity (an esterase activity, more specifically, glycerophosphodiester phosphodiesterase activity or the like) of OBDPF protein, a polypeptide containing a portion corresponding to this region can be used.
As a region corresponding to the fifth loop of the OBDPF protein, there may be mentioned, for example, a region comprising amino acid residues of about the 206th to 445th in mouse SEQ ID NO: 2 and a region comprising amino acid residues of about the 205th to 444th in human SEQ ID NO: 4. Among them, a portion responsible for the enzyme activity is in a region comprising amino acid residues of about the 225th to 328th in SEQ ID NO: 2, a region comprising amino acid residues of about the 224th to 327th in SEQ ID NO: 4 or in a region containing a proximate part thereof.
The enzyme activity can be measured and detected using a method, for example, described in a reference (Munson et al, J. Bacteriol., vol. 175, pp. 4569–4571, 1993), using glycerophosphocoline, glycerophosphoethanol-amine, etc. as a substrate.
Remodeling process of bone is as follows [References *, ** mentioned below.]. Initially, when lining cells of osteoblastic lineage at the resting stage which covers the bone surface are exposed to bone resorbing factors, such as PTH (parathyroid hormone), etc., these cells are activated to retract, whose morphology will change from flat epithelial-like cells to rounded cells. This causes a collagen matrix layer at the bone surface to be exposed. And the activated osteoblasts secrete collagenase, which dissolves the collagen matrix layer, whereby a bone mineral layer underneath is exposed.
Subsequently, these activated osteoblasts recruit precursors of the osteoclasts (that is, pre-osteoclasts) by means of cellular or hormonal signals.
Thus, when the osteoblasts directly contact with the pre-osteoclasts, a signal of ODF (osteoclast differentiation factor) (also referred to as RANKL (Receptor activator of NF-kb ligand)) expressed on the activated osteoblasts is transduced to the pre-osteoclasts, which is then differentiated into mature osteoclasts.
Next, these osteoclasts absorb bone. Subsequently, the osteoblasts secrete matrix proteins (such as collagen, osteocalcin, etc.), to form nodule, and thereby forming a collagen woven bone, called osteoid, and then, calcification occurs on the matrix of the osteoid, to give a newly formed bone. The osteoblast which completed their mission become the lining cells again to cover the bone surface.
The protein of the present invention is specifically expressed in bone tissue, and induces morphological change of the cells (more specifically, retraction), based on its esterase activity (more specifically, glycerophosphodiester phosphodiesterase activity or the like). From this fact, it is thought that the protein of the present invention plays an important role in an activation process of the osteoblasts (lining cells of osteoblastic lineage) and induction of successive recruitment of the osteoclasts, etc. in bone remodeling.
Therefore, as the function of the protein of the present invention in the osteoblasts or in a living organism which comprises the same, in addition to the above-mentioned functions (i), (ii) and (iii), there are included functions of inducing downstream phenomena in the process of bone remodeling.
Examples of such downstream phenomena include

    
    
        Secretion of collagenase from osteoblasts (*) (**)
        Dissolution of collagen matrix layer at a bone surface (*) (**)
        Recruitment of pre-osteoclasts (*)
        Increased expression of ODF (RANKL) on the cell surface of osteoblasts (*)
        Signal transduction of ODF (RANKL) (*)
        Differentiation of pre-osteoclasts to mature osteoclasts (*)
        Secretion of matrix proteins from osteoblasts (**)
        Nodule formation by osteoblasts (**)
    
    


Further, in case of detecting a function or an activity of the protein of the present invention in osteoblasts, the above-mentioned downstream phenomena in the bone remodeling process may be detected in stead of detecting a function or an activity of the above-mentioned (i), (ii) and (iii).
REFERENCES
* Manolagas S. C., Endocrine Reviews, 21(2): 115–137, 2000
** G. Gronowicz and L. g. Raisz, Bone Formation Assays, in “Principles of Bone Biology” (J. P. Bilezikian, L. G. Raisz, G. A. Rodan, eds.) Chapter 91, pp. 1253–1265, 1996, Academic Press Inc., USA.
The protein or the polypeptide of the present invention can be produced by overexpression by means of a usual genetic recombinant technology. In addition, they can be expressed and produced in a form of a fusion protein with other protein or polypeptide.
For example, a DNA encoding the protein is inserted in a vector in a way that it is operably jointed to a downstream of an appropriate promoter, thereby constructing an expression vector. Subsequently, the obtained expression vector is introduced in a host cell.
For an expression system (host cell-vector system), for example, there may be mentioned an expression system of bacteria, yeast, insect cells and mammalian cells. Among them, in order to obtain a protein having a well-reserved function, it is preferred to use insect cells (Spodoptera frugiperda SF9, SF21, etc.) and mammalian cells (Monkey COS-7 cell, Chinese hamster CHO cell, human HeLa cell, etc.) as a host cell.
For a vector, in case of a mammalian cell system, retrovirus type vector, papilloma virus vector, vaccinia virus vector, SV40 type vector, etc. can be used and in case of an insect cell system, bacurovirus vector, etc. can be used.
As a promoter, in case of the mammalian cell system, SV 40 promoter, LTR promoter, elongation 1α promoter, etc. can be used and in case of the insect cell system, polyhedrin promoter, etc. can be used.
As a DNA which encodes a protein or a polypeptide, cDNA corresponding to naturally existing mRNA (for example, those having a nucleotide sequence shown by SEQ ID NO: 1 and NO: 3) can be used, but it is not limited to those. It is possible to design a DNA that corresponds to an amino acid sequence of the desired protein, and to use it. In this case, 1 to 6 kinds of codons are known to encode one amino acid, respectively. Although a selection of the codon to be used may be voluntarily decided, by considering a codon frequency in a host cell employed for expression, it is possible to design a sequence with a higher expression efficiency. A DNA having a designed sequence can be obtained through chemical synthesis of DNA, fragmentation and combination of the above-mentioned cDNA, partial modification of a nucleotide sequence, and so on. Artificial modification of the nucleotide sequence in a part or mutagenesis can be carried out by a site specific mutagenesis (Proceedings of National Academy of Sciences, vol. 81, pp. 5662–5666, 1984) etc., using a primer comprising a synthesized oligonucleotide that encodes a desired modification.
The protein or the polypeptide of the present invention can be separated and purified by optionally combining conventional purification methods (salting out using inorganic salts, fractionating precipitation using an organic solvent, ion-exchange resin column chromatography, affinity column chromatography, gel filtration method, and so on).
A nucleic acid (an oligonucleotide or a polynucleotide) which is hybridizable with the gene or the nucleic acid of the present invention under stringent condition can be used as a probe for detecting the gene of the present invention. In addition, it may be used, for example, as an antisense oligonucleotide, a ribozyme or a decoy in order to modulate gene expression or function. Examples of such a nucleic acid may include a nucleotide comprising a partial sequence of consecutive 14 bases or more, or a complementary sequence thereof in nucleotide sequences shown in SEQ ID NO: 1, NO: 3, and NO: 5 to NO: 10.
Using the protein or the polypeptide (or the gene or the nucleic acid) and the-method for detecting the function or the activity (or the expression) thereof, etc. of the present invention, substances to be tested can be studied with respect to the effect on the function or activity of the protein or the polypeptide (or the gene or the nucleic acid) of the present invention.
Through these methods, it is possible to screen or identify compounds having an effect of modulating (inhibiting or enhancing) the function or the activity (or the expression) of the protein or the polypeptide (or the gene or the nucleic acid) of the present invention. The method for screening or identifying these compounds is thought to be useful in selecting or identifying pharmaceutical compounds (for example, therapeutic or prophylactic agent for diseases relating to bone metabolism disorders), which are valuable for selling.
An effect of the test substance on an expression of the gene of the present invention can be tested as follows. For example, a vector is constructed comprising a construct in which a regulatory region (a region containing a promoter, enhancer and soon) located in the 5′ upstream of the genomic DNA are connected with an appropriate reporter gene (for example, β-galactosidase gene, luciferase gene, etc.). And then, the vector is introduced into an appropriate cell. The cell is cultured in the presence of the test substance and an effect of the test substance on a gene expression is detected using an expression of the reporter gene as an index.
In addition, an effect of the test substance on the function or the activity of the protein or the polypeptide of the present invention can be detected as follows. For example, a test substance is brought in contact with cells expressing the protein or the polypeptide of the present invention, and a function or an activity of the protein or the polypeptide is detected. In comparison with a result obtained in the absence of the test substance, it can be determined whether or not the test substance has an effect of modulating the function or the activity, or a degree of the modulation effect. By using cells with no or less amount of expression of the protein or the gene of the present invention for a control, more accurate detection is possible. Further, in case of focusing on an enzyme activity (an esterase activity) as a function or an activity, isolated and purified protein or polypeptide or a part thereof of the present invention may be used in place of the cells. In this case, for example, a part containing an extracellular region, a region responsible for an enzyme activity, etc. may be used as such or in the form of a fused protein comprising these regions and other polypeptides.
From such a test result, screening, identification, evaluation, etc. can be carried out for an agent that modulates (inhibits or enhances) the function (or expression) of the protein or the polypeptide (or gene or nucleic acid) of the present invention.
When the protein of the present invention or an immunologically equivalent protein or polypeptide (synthesized polypeptide comprising a fragment of the protein or a partial sequence, etc.) is used as an antigen, an antibody can be obtained that recognizes the protein of the present invention. Immunologically equivalent protein means it causes cross reaction with an antibody for the protein of the present invention.
Polyclonal antibody can be produced by a conventional method by inoculating an antigen to a host animal (for example, rat, rabbit, etc.) and collecting an immunized serum. Monoclonal antibody can be produced by a technique such as a conventional hybridoma method. Further, by modifying the gene of the monoclonal antibody, humanized monoclonal antibody, etc. can be produced.
Using the above-obtained antibody, by an usual immunochemical method (such as enzyme immuno assay, etc.), an expression of the protein or the polypeptide of the present invention in cells or in tissues can be detected. Or else, by means of an affinity chromatography using an antibody, the protein of the present invention can be purified. Further, by using a neutralizing antibody, the function or the activity of the protein or the polypeptide of the present invention can be modulated.
Hereinbelow, the present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the scope of the present invention.
In Examples described below, each operation is conducted, unless otherwise specifically mentioned, according to a method described in “Molecular Cloning” (written by Sambrook, J., Fritsch, E. F. and Maniatis, T., published by Cold Spring Harbor Laboratory Press, 1989) or according to the instructions provided with the commercially available agents or kits.
EXAMPLES
Example 1
Isolation of cDNA of Mouse OBDPF Gene
1) Culture of Mouse Osteoblast-Like Cell Line MC3T3-E1
A mouse osteoblast-like cell line MC3T3-E1 (RIKEN RCB No.1126) was cultured as follows.
Cells (undifferentiated cells) were subcultured using α-MEM culture media (available from Gibco Co.) containing 10% bovine fetal serum. In case of having cells differentiated (matured) and calcified, the cells were cultured until confluent in the above-mentioned culture media, and ascorbic acid (0.2 mM) and β-glycerol phosphate (10 mM) were added to the culture media for inducing differentiation, and the mixture was further cultured for 11 to 14 days to obtain differentiated cells.
2) Isolation of a Gene whose Expression is Promoted at a Differentiation Stage of MC3T3-E1 into Osteoblasts
From undifferentiated cells of MC3T3-El which had been cultured in the same manner as in the above 1), and from the differentiated cells (cells cultured for 11 days after addition of ascorbic acid and β-glycerol phosphate) (each about 109 cells), total RNA was extracted, and mRNAs were purified using a mRNA separator kit (available from Clonetech Co.). Using the obtained mRNAs and according to differential display method (Liang et al., Science, vol. 257, pp. 967–971, 1992), candidate clones were selected as follows.
Using a reverse transcriptase and oligo (dT) primer, a single stranded cDNA was synthesized from the mRNA. Subsequently, PCR was carried out using the obtained single stranded cDNA. As the PCR primer, random primers (a primer of about 20 nucleotides size, comprising a random sequence) were used. The reaction was repeated for 4 cycles under conditions of at 95° C. for 40 seconds, at 30° C. for 1 minute, and at 72° C. for 1 minute, 30 cycles under conditions of at 95° C. for 40 seconds, at 55° C. for 1 minute, and at 72° C. for 1 minute, and for 1 cycle as the finalizing cycle, under conditions of at 72° C. for 5 minutes.
Such PCR reaction were carried out with respect to about 300 kinds of primers, and the obtained PCR products were applied to polyacrylamide gel electrophoresis, and stained with ethidium bromide. The bands developed on the gel ware observed and those identified in the sample derived from the differentiated cells and not identified in the sample derived from the undifferentiated cells were selected as candidate clones.
From the bands of the candidate clones, cDNA fragments were collected by elution, and amplified by once again carrying out PCR using the same primers. Subsequently, the amplified DNA was subcloned into a vector plasmid pGEM-T (available from Promega Co.).
3) Gene Expression in the Differentiated and Undifferentiated MC3T3-E1 Cells
Gene expression of the candidate gene in the differentiated and undifferentiated MC3T3-E1 cells were studied by Northern blotting. That is, Northern blotting was carried out using total RNA derived from the undifferentiated cells and the differentiated cells, and the cDNA fragments of the above-mentioned candidate clones as a probe. As a result, in the undifferentiated cells, no gene expression corresponding to the candidate clone was admitted, while in the differentiated cells, the expression was detected. Since the specific expression of the gene was confirmed in the differentiated cells, the candidate clone was thought to be a cDNA of a gene relating to bone metabolism.
4) Cloning of the cDNA and Determination of Nucleotide Sequence
From the mRNA derived from the differentiated cells obtained in the same manner as in the above 2), cDNA library was prepared. Using the cDNA fragments (α-32P-dCTP labeled) of the candidate clone obtained in the above 2) as a probe, plaque hybridization was carried out under highly stringent conditions, with respect to the above-mentioned cDNA library.
Among the positive clones, those with a longer insertion fragment was selected and with respect to plasmids derived from these clones, various kinds of deletion plasmids were prepared and nucleotide sequence of the inserted cDNA was determined by the dideoxy method. The cDNAs whose nucleotide sequences were determined were linked to obtain the whole cDNA of mouse OBDPF gene.
Through analysis on the nucleotide sequence of the cDNA, an open reading frame was identified, and then, an amino acid sequence of the protein encoded thereby was determined. The nucleotide sequence of the whole cDNA was shown in SEQ ID NO: 1, and the amino acid sequence of the OBDPF protein encoded thereby was shown in SEQ ID NO: 2 in the sequence listing mentioned below.
The molecular weight of the mouse OBDPF protein presumed from the amino acid sequence was about 61 Kd. Further, from the analysis on hydropathy of this amino acid sequence, OBDPF protein is expected to have a membrane protein like structure containing 7 transmembrane domains. It is also thought to have one leucine zipper and about eight N-glycosilation regions. Schematic drawing of the expected structure is shown in FIG. 1.
Example 2
Isolation of cDNA of Human OBDPF Gene
The fragment comprising a coding region of the whole cDNA of the mouse OBDPF gene was labeled with α-32P-dCTP, and using this as a probe, plaque hybridization was carried out with respect to human spleen cDNA library (available from Stratagene Co.), to obtain positive clones. Plaque hybridization was carried out under normal stringent condition.
Among the obtained positive clones, those with a longer insertion fragment was selected and with respect to plasmids derived from these clones, nucleotide sequence of the inserted cDNA was determined in the same manner as in the above examples 1–4).
Thus, the whole cDNA of the human OBDPF gene was obtained. Through analysis on the nucleotide sequence of the cDNA, an open reading frame was identified, and then, an amino acid sequence of the protein encoded thereby was determined. The nucleotide sequence of the whole cDNA was shown in SEQ ID NO: 3, and the amino acid sequence of the OBDPF protein encoded thereby was shown in SEQ ID NO: 4 in the sequence listing below.
The molecular weight of the human OBDPF protein presumed from the amino acid sequence was about 61 Kd. Further, from a comparison between the mouse and human sequences, homology between the nucleotide sequences of cDNAs of the mouse and human OBDPF genes was about 87% in the coding region (about 1.6 kb). In addition, homology between the amino acid sequences of the mouse and human OBDPF protein (539 amino acid residues) was about 87%.
Example 3
Isolation of Genomic Gene of the Mouse OBDPF
Using the whole cDNA fragment of the mouse OBDPF gene obtained in Example 1 as a probe, plaque hybridization was carried out with respect to mouse SvJ genomic library (available from Stratagene Co.), to obtain positive clones containing DNA of genomic OBDPF gene. The genomic DNA portion of these positive clones were subcloned in a vector plasmid pBluescript (available from Stratagene Co.). Subsequently, by means of Southern blotting, cDNA region (exon region) existing on a genomic DNA of each clone was determined, and nucleotide sequence of the DNA was determined by dideoxy method, with respect to the clones containing the exon region. By comparing the nucleotide sequences of the genomic DNA and the whole cDNA of the OBDPF gene, exon-intron existing region was identified.
In SEQ ID NO: 5 to 12 of the sequence listing below, the nucleotide sequences of exon-intron boundary region of the mouse OBDPF genomic DNA were shown. Each of the SEQ ID NO: 5, NO: 6, NO: 7, NO: 8, NO: 9, NO: 10, NO: 11 and NO: 12 shows, respectively, a nucleotide sequence of exon 1, exon 2, exon 3, exon 4, exons 5 to 10, exon 11, exon 12 and exons 13 to 16, and nucleotide sequences of introns spacing at the both ends of each exon. In addition, restriction enzyme map of the mouse OBDPF genomic DNA and a location of the exons were shown in FIG. 2.
Example 4
Expression of the OBDPF Gene in a Differentiation Process of Osteoblasts
Mouse osteoblast-like cell line MC3T3-E1 was induced to differentiate by culturing the cells in a culture media to which ascorbic acid and β-glycerol phosphate were added in the same manner as in Example 1, and total RNA was prepared from the cells after 0, 4, 7, 11 and 15 days of culture. Using the Pst I fragment of the cDNA of the mouse OBDPF gene obtained in Examples 1–3) (a fragment corresponding to 662th to 1112th base of the SEQ ID NO: 1) as a probe, Northern blotting was carried out with respect to these total RNAs.
As a result, as shown in FIG. 3, expression of mRNA of OBDPF was hardly detected after 0 days of culture (right after confluent) (in lane 1), while significant increase in expression were observed after 4 and 7 days (in lanes 2 and 3). Beyond that, after 11 and 15 days (in lanes 4 and 5), expressions were slightly decreased, however, an expression level was still high in comparison with 0 day culture. As shown above, since the expression amount changed in accordance with a progress of differentiation (maturation) of MC3T3-E1 cells, OBDPF gene was thought to be involved in a differentiation (maturation) of the osteoblasts.
Example 5
Expression of the OBDPF Gene in Various Kinds of Tissues in Mouse
Expression pattern of the OBDPF gene in various tissues (heart, brain, spleen, lung, liver, skeletal muscle, kidney, testis, femur, and calvaria) was studied by Northern blotting as follows.
Tissues from femur and calvaria were collected from a mouse (ICR line male mouse, 12 weeks old) and poly (A)+RNA was prepared. For other tissues, commercially available mouse poly (A)+RNA (prepared from BALB/c line mouse; trade name, mouse MTN blots, available from Clonetech Co.) were used.
Northern blotting was carried out with respect to poly (A)+RNAs derived from each of the above-mentioned tissues. As a probe, Pst I fragment of cDNA of the mouse OBDPF gene obtained in Examples 1–3) (corresponding to 662th to 1112th nucleotide sequence of the SEQ ID NO: 1) was used.
As a result of the Northern blotting, as shown in FIG. 4, bands were detected of about 2.5 kb in femur, calvaria and spleen, and expressions were confirmed.
Example 6
Functional Analysis of OBDPF (I)
(Induction of Differentiation of Osteoblasts by Expression of OBDPF)
1) Construction of an OBDPF Expression Vector and Preparation of Cells Over-Expressing OBDPF
A BLUNT-ENDED cDNA fragment of the mouse OBDPF gene obtained in Examples 1–3) (a fragment corresponding to from the 197th to the 1851th base of SEQ ID NO: 1, containing entire coding region) was ligated in a vector plasmid containing elongation 1 α promoter, downstream of the above-mentioned promoter (Spe I restriction site) in a reading direction, to construct an OBDPF expression vector.
The above-obtained OBDPF expression vector was made linear by a restriction enzyme Pvu I. This was introduced into mouse osteoblast-like cell line MC3T3-E1 by means of electropolation method, and the cells were cultured in an α-MEM culture media containing neomycin (G418) for 10 days. Subsequently, 10 clones which were resistant to G418 were selected. These G418 resistant cells (that is, cells into which the expression vectors were introduced) were subjected to RT-PCR (reverse transcript-polymerase chain reaction), thereby to confirm overexpression of OBDPF mRNAs.
2) Analysis on Differentiation Marker of Osteoblasts in Cells Overexpressing OBDPF
The cells overexpressing OBDPF from the above-obtained 10 clones (referred to as S01, S02, S05, S06, S07, S09, S13, S15, S17 and S18) were cultured until confluent in the culture media, andascorbic acid (0.2 mM) and β-glycerolphosphate (10 mM) were added to the culture media for inducing differentiation, and the mixture was cultured for 0 to 14 days, and then, an alkaline phosphatase activity and calcium deposition amount were measured. As a control were used cells into which vector was introduced.
The alkaline phosphatase activity was measured as follows. The cells were washed with PBS (phosphate buffered saline), and suspended in 50 mTris-HCl (pH 7.5) containing 0.1% Triton-100, and lysed ultrasonically to obtain an enzyme solution. Using a kit for measuring phosphatase activity (Phosphatase Substrate System, available from Kirkegaard & Perry Laboratories Co.), an activity was measured using p-nitrophenyl-phosphate as a substrate.
Calcium deposition amount was measured as follows. The cells were washed with PBS (phosphate buffered saline), and dissolved with 0.5N hydrochloric acid. After overnight treatment at 4° C., centrifugation was carried out to obtain a supernatant, and a calcium amount in the supernatant was measured using s kit for measuring calcium content (Calcium C Test Wako, available from Wako Junyaku Co.), according to Orthocresol phthalane Complexon method (OCPC method).
The results are shown in FIG. 5. The alkaline phosphatase activity after 0 to 14 days culture is shown in (A) and the calcium deposition amount after 14 days culture is shown in (B), respectively.
As shown in FIG. 5, in the cells overexpressing OBDPF, significant increases were confirmed in both of the alkaline phosphatase activity and the calcium deposition amount, as compared to the control cells (the cells into which vectors were introduced). Thus, since the overexpression of OBDPF resulted in a significant increase in the marker of differentiation in osteoblasts and active bone formation potential, it was concluded that OBDPF had an effect of promoting differentiation (maturation) of osteoblasts.
Example 7
Functional Analysis on OBDPF (II)
(Enzymatic Function of OBDPF and Morphological Change in Cells by OBDPF Expression)
1) Construction of a Green Fluorescent Protein (GFP) Fused OBDPF Expression Vector
The cDNA fragment of OBDPF obtained in Examples 1–3) (a fragment corresponding to the 16th to the 1821th base of SEQ ID NO: 1; containing an entire coding region but not containing a stop codon) was ligated to Xho I and BamH I restriction sites of pEGFP-N1 (available from Clonetech Co.), in a reading direction to construct an expression vector for expressing GFP fused with OBDPF.
2) Transient Expression in 293T Cells and Staining of Actin Filament
Using Lipofection method, the above-mentioned GFP fused OBDPF expression vector was introduced into 293T cells and it was overexpressed transiently, as follows.
Specifically, 1×105 of 293T cells (available from Dainihon Seiyaku Co.) were inoculated onto a culture slide (available from Falcon Co.) and cultured overnight. For culture media for the 293T cells, DMEM (available from Lifetech Co.) containing 10% bovine fetal serum was used. On the following day, 3 μg of the GFP fused OBDPF expression vector (as a control, pEGFP-N1 was used in place of this vector), (which had been dissolved in 100 μl of buffer (Opti-MEM; available from Lifetech Co.) and 6 μl of an agent for Lipofection (which had been dissolved in 100 μl of Opti-MEM) were mixed, and the mixture was incubated at room temperature for 15 minutes. Subsequently, this was added dropwise to the above-mentioned cell culture liquid and the mixture was cultured overnight. After the culture liquid was removed, the cells were fixed with a neutral phosphate buffer containing 4% paraformaldehyde and 4% sucrose at room temperature for 30 minutes, and washed with phosphate buffer for 3 times. Subsequently, added thereto was 1 ml of phosphate buffer containing rhodamine-labeled phalloidin, and the mixture was incubated at room temperature for 2 hours. The resultant mixture was washed with phosphate buffer for 3 times and mounted with phosphate buffer containing 50% glycerol. Using a microscope (BX-60; available from Olimpus Co.), fluorescence was observed and photographed by a digital camera (Sensys; available from Olimpus Co.).
As a result, in the cells overexpressing the wild type GFP by introducing pEGFP-N1, the wild type GFP was present in entire cytoplasm. On the other hand, in the cells overexpressing the GFP fused OBDPF by introducing the GFP fused OBDPF expression vector, the GFP fused OBDPF was localized in the peripheral part of the cells. Additionally, the cells overexpressing the GFP fused OBDPF changed their shapes to a spherical form and actin filaments disappeared.
3) Transient Expression of a Mutant OBDPF in 293T Cells
On the 5th loop of the OBDPF, which is an extracellular domain, there exists an amino acid sequence showing an extremely high homology with glycerophosphodiester phosphodiesterase (EC3.1.4.46) which has been reported in bacteria and yeasts.
This portion on the 5th loop of OBDPF corresponds to, for example, the 225th to the 328th amino acid residues in the mouse OBDPF (SEQ ID NO: 2) and the 224th to the 327th amino acid residues in the human OBDPF (SEQ ID NO: 4).
Particularly, the arginine residue at the 231th in the mouse OBDPF (the 230th in the human OBDPF) is well conserved in E. coli-derived 2 kinds of glycerophosphodiester phosphodiesterase (ecUGPQ, and ecGLPQ) and the same enzyme in Haemophilis influenzae (hiGLPQ) (see FIG. 6), it is expected to be essential for the activity.
In order to test this assumption, a mutant of the mouse OBDPF was prepared in which the arginine residue at the 231st was replaced with an alanine residue. Specifically, the GFP fused OBDPF expression vector, a synthesized DNA (available from Lifetech Co.), and a kit for site-directed mutagenesis (Quick Change Site-Directed Mutagenesis Kit; available from Stratagene Co.) were used to prepare a GFP fused mutant OBDPF expression vector where mutation was introduced.
(As the synthetic DNA, those having the following nucleotide sequence were used. 5′-GGG CTG GTG GGA CAC GCA GGG GCC CCC ATG CTG-3′ (SEQ ID NO: 18) 5′-CAG CAT GGG GGC CCC TGC GTG TCC CAC CAG CCC-3′) (SEQ ID NO: 19)
The obtained GFP fused mutant OBDPF expression vector was introduced into 293T cells by Lipofection method, and it was overexpressed transiently.
As a result, when a localization in the cell was studied, the GFP fused mutant OBDPF was localized in the peripheral part of the cell as is the case for the GFP fused OBDPF (wild type). However, with respect to the morphology of the cell, no morphological change was observed when the GFP fused mutant OBDPF was overexpressed, while those overexpressing the wild type changed their shapes to a spherical form.
4) About the Function of OBDPF
From the results of the above 1) to 3), it is expected that the OBDPF protein has an esterase activity (glycerophosphodiester phosphodiesterase activity), and a portion on the 5th loop which is an extracellular part is responsible for this enzymatic activity.
Further, it was shown that expression of the OBDPF induced morphological change of a cell (retraction). Further, from the result of the mutagenesis, a function of inducing such morphological change (retraction) is based on the above-mentioned enzyme activity.
From the above facts and other characteristics (that is, the fact that OBDPF is expressed specifically in bone tissues, and the fact that it is expressed specifically at a differentiation stage of osteoblasts), OBDPF is thought to have a function of inducing morphological change (retraction) of the osteoblasts, particularly. In addition, there is a possibility that the OBDPF exhibits an important function of inducing adhesion of the osteoclasts to a bone surface at an initial stage of bone absorption during a bone remodeling.
The protein, the polypeptide, the gene, or the nucleic acid and the method of detecting a function or an activity thereof of the present invention are useful in elucidating a mechanism of bone metabolism, especially, differentiation of the osteoblasts and bone remodeling.
Further, they are useful in studies on pathological states, diagnostics, therapeutic and prophylactic treatment and research and development of pharmaceuticals for the diseases such as osteoporosis, osteopeterosis, osteomalacia, hypercalcemia, etc.

## Claims

1. A method of assaying a function or an activity of an osteoblast differentiation promoting factor (OBDPF) protein, said method comprising:
culturing a cell expressing an OBDPF protein, and
detecting a function or activity of said OBDPF protein in said cell;
wherein said cell is an osteoblast, and said function or activity is promoting differentiation of the osteoblast to change from an immature state to a mature state;
said OBDPF protein is a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or a polypeptide encoded by a nucleic acid which hybridizes under stringent conditions with a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3,
said hybridization being carried out by conducting hybridization for 16 hours in 6×SSC or a hybridization solution having an equivalent salt concentration at a temperature of 50 to 60° C., followed by preliminary washing with 6×SSC or a solution having an equivalent salt concentration as necessary, and subsequently washing with 1×SSC or a solution having an equivalent salt concentration.

2. The method of claim 1, wherein said function or activity is detected by a marker selected from i) an amount of calcification, ii) an alkaliphosophatase activity, iii) an osteocalcin activity, and iv) an amount of expression of osteopontin.

3. A method of assaying a function or an activity of an osteoblast differentiation promoting factor (OBDPF) protein, said method comprising:
culturing a cell expressing OBDPF protein, and
detecting a function or activity of said OBDPF protein in said cell;
wherein said function or activity is induction of retraction of said cell;
said OBDPF protein is a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or a polypeptide encoded by a nucleic acid which hybridizes under stringent conditions with a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3;
said hybridization being carried out by conducting hybridization for 16 hours in 6×SSC or a hybridization solution having an equivalent salt concentration at a temperature of 50 to 60° C., followed by preliminary washing with 6×SSC or a solution having an equivalent salt concentration as necessary, and subsequently washing with 1×SSC or a solution having an equivalent salt concentration.

4. The method of claim 3, wherein said cell is an osteoblast.

5. The method of claim 1 or 3, wherein said OBDPF protein is the polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

6. The method of claim 1 or 3, wherein said OBDPF protein is the polypeptide comprising the amino acid sequence of SEQ ID NO:4.

7. The method of claim 1 or 3, wherein said OBDPF protein is the polypeptide encoded by the nucleic acid which hybridizes under highly stringent conditions with the nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.

8. The method of claim 7, wherein said OBDPF is the polypeptide encoded by the nucleic acid which hybridizes under highly stringent conditions with the nucleic acid comprising the nucleotide sequence of SEQ ID NO:3.

9. The method of claim 1 or 3, said method further comprising introducing a nucleic acid encoding said OBDPF protein into said cell expressing the OBDPF protein.

10. The method of claim 1 or 3, wherein said OBDPF protein is isolated or recombinant.

11. The method of claim 1 or 3, wherein said OBDPF protein includes an extracellular region.