HK1123578A

HK1123578A - Polypeptides controlling phosphoric acid metabolism calcium metabolism calcification and vitamin d metabolism and dnas encoding the same

Info

Publication number: HK1123578A
Application number: HK09101223.8A
Authority: HK
Inventors: 山下武美; 岛田孝志; 水谷悟; 福本诚二
Original assignee: 麒麟医药株式会社
Priority date: 2000-08-11
Filing date: 2009-02-10
Publication date: 2009-06-19

Description

Polypeptides regulating phosphate metabolism, calcium metabolism, calcification and vitamin D metabolism and DNA encoding the same

The present application is a divisional application of an invention patent application having an application date of 2001, 8/10 and an application number of 01817296.2 and having the same invention title as the present invention.

Technical Field

The present invention relates to a polypeptide which regulates phosphate metabolism, calcium metabolism, calcification and/or vitamin D metabolism, a DNA encoding the polypeptide, and a pharmaceutical composition containing the polypeptide as an active ingredient, and an antibody recognizing the polypeptide, and a pharmaceutical composition using the antibody as an active ingredient, a diagnostic method and a diagnostic composition using the antibody.

Background

Inorganic phosphate (hereinafter also referred to as phosphate) is essential for energy metabolism and maintenance of cell function in vivo, and plays an important role in tissue calcification together with calcium. Phosphate supply to the organism is mainly determined by absorption in the intestine, while phosphate excretion is determined by urinary excretion in the kidney and fecal excretion in the intestine. In living organisms, phosphate is distributed in body fluids, intracellular components and calcified tissues. The levels of inorganic phosphate excretion and absorption in adults are nearly identical, indicating that a regulatory mechanism exists that maintains the homeostasis of phosphate metabolism. It is well known that calcium metabolism, which is similar to phosphate metabolism in its distribution and homeostatic control of blood levels, is synergistically regulated in mammals by regulatory factors, such as at least parathyroid hormone, calcitonin and 1 α, 25-dihydroxyvitamin D3.

In the regulation of phosphate metabolism, parathyroid hormone is known to promote the excretion of phosphate in the intestinal tract, and 1 α, 25-dihydroxyvitamin D3 promotes the absorption of phosphate in the intestinal tract. This clearly demonstrates a close relationship between phosphate metabolism and calcium metabolism. However, the main substances regulating phosphate have not been clarified so far.

Currently, diseases associated with loss of homeostasis of phosphate metabolism and low levels of inorganic phosphate in the blood include primary hyperparathyroidism, hereditary rickets from blood phosphate and tumor-induced osteomalacia.

Primary hyperparathyroidism is manifested as an excess of parathyroid hormone in the parathyroid gland, and we know that it can progress to hypophosphatemia with increased phosphate excretion, since excess parathyroid hormone inhibits the reabsorption of inorganic phosphate in the kidney.

In addition, examples of hypophosphatemia known to be caused by genetic diseases include vitamin D-dependent rickets type I, vitamin D-dependent rickets type II, and vitamin D-resistant rickets. Vitamin D-dependent rickets type I are diseases caused by genetic dysfunction of synthetase producing active vitamin D metabolites, and vitamin D-dependent rickets type II are diseases caused by genetic dysfunction of vitamin D receptors. Both diseases develop hypophosphatemia with hypocalcemia due to hypofunction of vitamin D3 metabolite. In contrast, for vitamin D resistant rickets, there are known at least two clinical symptoms of different causes, X-linked chromosomal and autosomal hypophosphatemia rickets.

The clinical symptoms of vitamin-resistant rickets mentioned above all lead to hypophosphatemia, characterized by renal phosphate. Recently, it was shown in X-linked rickets with hypophosphatemia (hereinafter referred to as XLH) that the disease is induced by mutation of a gene encoding a endopeptidase-like protein named PHEX located on the X chromosome. However, the mechanism of the dysfunctional PHEX protein to induce hypophosphatemia has not been elucidated. Interestingly, genetic analysis of a naturally occurring mutant mouse (Hyp) with hypophosphatemia revealed a partial deletion of the PHEX-encoding gene in this mouse. Experiments with these mice showed that PHEX deficient mice have normal renal function and that in the body fluid of Hyp mice there is a humoral factor which is different from parathyroid hormone but can also induce hypophosphatemia. For autosomal dominant rickets/osteomalacia (hereinafter referred to as ADHR), a gene causing the disease is being sought, and linkage analysis indicates that one such gene exists within the 12p13 region. However, the region which has been narrowed down is still too wide and contains many genes, and there is no clear candidate gene.

Tumor-induced osteomalacia can progress to hypophosphatemia associated with tumorigenesis, with increased renal phosphate, characterized by elimination of hypophosphatemia by irradiation or resection of the tumor. This disease is believed to produce a factor in the tumor that induces hypophosphatemia by inhibiting phosphate reabsorption in the kidney.

It has not been confirmed whether the molecule causing vitamin D resistant rickets is the same as the inducing molecule of tumor-induced osteomalacia. However, both molecules are apparently unknown phosphate metabolism factors that promote urinary phosphate excretion. Putative phosphate metabolism regulators are often referred to as Phosphatonin. The relationship of this unknown regulator of phosphate metabolism to vitamin D resistant rickets or tumor induced osteomalacia has been summarized as a summary (Neison, A.E., Clinical Endocrinology, 47: 635-642, 1997; Drezner, M.K., Kidney Int., 57: 9-18, 2000).

Another characteristic of vitamin D resistant rickets or tumor-induced osteomalacia is damage to bone calcification. Damaged bone calcification can be thought to be the result of secondary progression of hypophosphatemia. However, since abnormal skeletal calcification in experiments using Hyp mice, model mice for vitamin D resistant rickets showed development independent of phosphate levels (Ecarot, B., J.bone Miner. Res., 7: 215-220, 1992; Xiao, Z.S., am.J.physiol., E700-E708, 1998), it is possible that the above unknown regulatory factors of phosphate metabolism could directly regulate skeletal tissue calcification.

As mentioned above, the research data strongly suggest the presence of an unknown factor that regulates phosphate metabolism, but there are no examples that specify this entity with the desired activity at the molecular level. WO99/60017 discloses a novel polypeptide sequence of a novel polypeptide hormone (phatonin), but it does not disclose the unique activity of phatonin to induce hypophosphatemia. Thus, it is conceivable that an unidentified intrinsic factor regulating phosphate metabolism is present in the organism.

The vitamins D2 and D3 ingested from food, or vitamin D3 synthesized in the skin, are hydrolyzed by vitamin D-25-hydroxylase mainly present in the liver to produce 25-hydroxyvitamin D. The 25-hydroxyvitamin D is then hydrolyzed by 25-hydroxyvitamin D-1 alpha-hydroxylase present in the epidermal cells of the proximal tubule of the kidney to produce 1 alpha, 25-dihydroxyvitamin D. This substance is a physiologically active mineral regulatory hormone which increases serum calcium and phosphate levels and is known to inhibit parathyroid hormone secretion and to participate in the promotion of bone resorption. Then, 1 α, 25-dihydroxyvitamin D is converted by 24-hydroxylase, which is mainly present in the kidney and small intestine, into a metabolite that does not have the above-mentioned physiological activity. In this regard, 24-hydroxylase is considered to be an enzyme that inactivates 1 α, 25-hydroxy vitamin D. On the other hand, 24-hydroxylase is also known to act on 25-hydroxy vitamin D, converting it to 24, 25-dihydroxy vitamin D. 24, 25-dihydroxyvitamin D has been reported to have physiological effects of increasing bone mass or promoting cartilage differentiation, suggesting that this enzyme also has aspects of producing bioactive vitamin D metabolites.

Known factors that regulate the expression level of 1 α -hydroxylase, which plays an important role in activation of vitamin D, include parathyroid hormone (PTH), calcitonin, 1 α, 25-dihydroxy vitamin D, and the like. The decrease in calcium content in blood promotes the secretion of PTH, which acts on PTH receptors present in the epidermal cells of the proximal tubule of the kidney to promote the transcription of 1 α -hydroxylase by increasing the intracellular cAMP level, resulting in an increase in the concentration of 1 α, 25-dihydroxyvitamin D in blood. 1 alpha, 25-dihydroxyvitamin D promotes the absorption of calcium in the intestinal tract and the reabsorption of calcium in the kidney, thereby increasing the calcium content in the blood. In addition, it has been reported that 1 α, 25-dihydroxyvitamin D binding to Vitamin D Receptor (VDR) acts on the promoter region of 1 α -hydroxylase gene or PTH gene to inhibit transcription of these genes. Specifically, 1 α, 25-dihydroxyvitamin D has a feedback regulatory mechanism for its activating factors, PTH and 1 α -hydroxylase. This mechanism plays an important role in maintaining calcium metabolism homeostasis.

Recently, it has been reported that a decrease in serum phosphate levels enhances the expression of the 1 α -hydroxylase gene. In phosphate metabolism, it is also estimated that there is a mechanism whereby an increase in 1 α -hydroxylase expression associated with a decrease in serum phosphate levels can increase serum 1 α, 25-dihydroxyvitamin D levels, thereby correcting serum phosphate levels by promoting phosphate absorption in the small intestine.

Factors responsible for regulating the expression of the 24-hydroxylase gene include 1 α, 25-dihydroxyvitamin D and PTH. 1 α, 25-dihydroxyvitamin D interacts with Vitamin D Receptor (VDR), and the complex binds to a vitamin D receptor response sequence present in the promoter region of the 24-hydroxylase gene to promote transcription. 1 α, 25-dihydroxyvitamin D is believed to activate 24-hydroxylase and then induce a decrease in the level of 1 α, 25-dihydroxyvitamin D as a result of the metabolic pathway being activated. It is known that the expression of the 24-hydroxylase gene is inhibited by PTH, but the detailed molecular mechanism thereof is not clear.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a novel tumor derived factor which induces a decrease in blood phosphate levels.

It is envisaged that tumours identified in tumour-induced osteomalacia will secrete a physiologically active soluble factor which results in a decrease in blood phosphate levels. This tumor-derived factor destabilizes phosphate metabolism. Therefore, the factor can be classified into one of (1) the factor is not produced in vivo and is produced specifically in a tumor, (2) the factor is produced in an excessive amount in a tumor, although it is also produced in a normal tissue, and (3) the factor is produced in a tumor without being physiologically controlled.

Based on the hypothesis that the hypophosphatemia-induced tumor-derived factor as described above characteristically develops in tumor-induced hypophosphatemia-derived tumors, we expect enhanced transcription of the factor encoding the hypophosphatemia-inducing factor or enhanced stability of the mRNA of the factor in the tumor. Therefore, a part of tumor tissue was excised from a tumor-induced hypophosphatemia patient for therapeutic purposes, and after extracting RNA therefrom, a cDNA library was prepared using a phage vector and a plasmid vector, and then a gene fragment specifically expressed in a tumor was screened. The screening method is to select cDNA fragments specifically expressed in the tumor and to select cDNA fragments in the tumor-derived cDNA library that do not cross-react with cDNA probes from the kidney proximal tubule epidermal cell line. We further narrowed the selected cDNA fragments by verifying the novelty of the sequence and the specific expression in tumors, thereby obtaining cDNA fragments expected to encode the hypophosphatemia inducing factor. From the sequence information, we attempted to clone the cDNA containing the ORF where each fragment is located, and successfully obtained DNA encoding the novel polypeptide. We further studied the biological activity of these novel polypeptides thoroughly, so we completed the present invention by elucidating that the novel polypeptides have activities of inhibiting phosphate transport, inducing hypophosphatemia and inhibiting calcification of bone tissue in animals.

Specifically, the present invention is as follows.

(1) A DNA encoding the following polypeptide (a), (b), (c) or (d):

(a) consisting of SEQ ID NO: 2 or 4, or a pharmaceutically acceptable salt thereof

(b) Consisting of SEQ ID NO: 2 or 4 by deletion, substitution or addition of 1or several amino acids, and the polypeptide has hypophosphatemia inducing activity, phosphate transport inhibiting activity, calcification inhibiting activity or activity for regulating vitamin D metabolism in vivo,

(c) consisting of SEQ ID NO: 2, wherein the partial sequence contains at least the 34 th to 201 th amino acids in the amino acid sequence, or

(d) Consisting of SEQ ID NO: 2, wherein the partial sequence:

(i) an amino acid sequence containing from the 34 th to 201 th amino acids in the above amino acid sequence,

(ii) consisting of an amino acid sequence obtained by deletion, substitution or addition of 1or several amino acids of said partial sequence, and

(iii) has hypophosphatemia inducing activity, phosphate transport inhibiting activity, calcification inhibiting activity or in vivo vitamin D metabolism regulating activity.

(2) A DNA comprising the following DNA (e) or (f):

(e) consisting of SEQ ID NO: 1 from nucleotide 133 to 885,

or by SEQ ID NO: 3 from nucleotide 1 to 681 in the nucleotide sequence shown in the above, or

(f) DNA which hybridizes under stringent conditions to a probe consisting of the nucleotide sequence of SEQ ID NO: 1or 3, or a DNA consisting of all or part of the nucleotide sequence shown in the sequence ID No.

Here, the term "stringent conditions" satisfies the conditions that the salt concentration is 750mM or more, preferably 900mM or more, and the temperature is 40 ℃ or more, preferably 42 ℃. Specifically, stringent conditions consist of 6 XSSC, 5 XDenhardt, 0.5% SDS, 50% formaldehyde and 42 ℃.

(3) A recombinant vector containing the above DNA.

(4) A transformant containing the recombinant vector.

(5) A polypeptide which is a polypeptide (a), (b), (c) or (d)

(a) Consisting of SEQ ID NO: 2 or 4, or a pharmaceutically acceptable salt thereof,

(b) consisting of SEQ ID NO: 2 or 4 by deletion, substitution or addition of 1or several amino acids, which has hypophosphatemia-inducing activity, phosphate transport-inhibiting activity, calcification-inhibiting activity or in vivo vitamin D metabolism-regulating activity,

(c) consisting of SEQ ID NO: 2, wherein said partial sequence comprises an amino acid sequence of at least amino acids 34 to 201 of the above sequence, or

(d) Consisting of SEQ ID NO: 2, wherein the partial sequence:

(i) an amino acid sequence comprising at least amino acids 34 to 201 of the above sequence,

(ii) an amino acid sequence obtained by deleting, substituting or adding one or more amino acids from the partial sequence, and

The above-mentioned polypeptide also includes a polypeptide modified with at least one substance selected from the group consisting of polyethylene glycol, dextran, poly (N-vinyl-pyrrolidone), polypropylene glycol homopolymer, copolymer of polypropylene oxide and polyethylene oxide, polyoxyethylated polyol and polyvinyl alcohol.

(6) A pharmaceutical composition containing the above polypeptide as an active ingredient.

The pharmaceutical composition can be used for regulating calcium metabolism, phosphate metabolism, calcification or vitamin D metabolism in vivo. In addition, the pharmaceutical composition is effective against at least one symptom of hyperphosphatemia, hyperparathyroidism, renal osteodystrophy, ectopic calcification, osteoporosis, and vitamin D hyperactivity.

(7) An antibody reactive with the above polypeptide or a partial fragment thereof.

The antibody can be obtained by a method comprising the step of immunizing an animal with the polypeptide of the present invention or a partial fragment thereof as an antigen.

(8) A pharmaceutical composition containing the above antibody as an active ingredient.

The pharmaceutical composition can regulate calcium metabolism, phosphate metabolism, calcification or vitamin D metabolism in vivo, or effectively resist bone diseases. Here, the bone disease is at least one disease of osteoporosis, vitamin D-resistant rickets, renal osteodystrophy, dialysis-related bone disease, calcified insufficiency bone disease, paget's disease, and tumor-induced osteomalacia.

(9) A diagnostic reagent comprising the above antibody for a disease exhibiting abnormality in at least one of abnormal calcium metabolism, abnormal phosphate metabolism, abnormal calcification and abnormal vitamin D metabolism (for example, one of renal failure, renal phosphate leakage, renal tubular acidosis and fanconi syndrome).

(10) A diagnostic reagent for bone diseases comprising the above antibody, wherein the bone diseases are at least one of osteoporosis, vitamin D-resistant rickets, renal osteodystrophy, dialysis-related bone diseases, calcified insufficiency bone diseases, paget's disease and tumor-induced osteomalacia.

(11) A diagnostic reagent comprising a peptide having SEQ ID NO: 11or a partial fragment thereof, for use in a disease exhibiting an abnormality in at least one of abnormal calcium metabolism, abnormal phosphate metabolism, abnormal calcification and abnormal vitamin D metabolism.

An example of the above partial sequence has SEQ ID NO: 11, from nucleotide 498 to 12966. An example of such a disease is autosomal dominant rickets/osteomalacia.

The present invention is explained in detail as follows. This specification includes the contents of the specification and/or drawings of Japanese patent applications 2000-245254, 2000-287684, 2000-391077 and 2001-121527, which are the priority documents of the present application.

The terms used in the present specification are defined as follows.

The term "activity to reduce the level of blood 1, 25-dihydroxyvitamin D3" means an activity to produce an effect of reducing the level of blood 1, 25-dihydroxyvitamin D3.

The term "hypophosphatemia-inducing activity" means an activity of reducing the level of phosphate in blood. Blood phosphate levels are defined by the balance between (i) absorption and excretion to the intestine into urine and feces, and (ii) the distribution of phosphate in cells in the body and in calcified tissues, represented by bone tissue. Thus, the term "hypophosphatemia-inducing activity" as used in this specification means an activity of reducing the blood phosphate level in a healthy living body, and does not necessarily refer to an activity of causing pathological hypophosphatemia. At the tissue level, the hypophosphatemia-inducing activity may be equivalent to phosphate absorption inhibitory activity in the intestine, phosphate excretion promoting activity in the kidney or intestine, or activity to promote phosphate transfer to cells.

Further, the term "phosphate transport inhibitory activity" in the present invention means an activity of acting on a target cell to inhibit the activity of a phosphate transport carrier present on the cell membrane. The possible target cells are mainly epidermal cells of the renal duct, of the intestine or of osteoblasts.

In addition, the term "calcification suppressing activity" in the present invention means an activity of suppressing generation or accumulation of a crystalline substance containing calcium and phosphate as a component of bone tissue and soft tissue.

In addition, the term "in vivo vitamin D metabolism-regulating activity" means the ability to regulate the change in absolute amount or abundance of vitamin D present in the body and metabolites synthesized from it in the body. In vivo regulation of vitamin D and its metabolites is controlled primarily by (i) absorption or excretion in the gut and (ii) reabsorption or excretion in the kidney, as well as (iii) in vivo synthesis of vitamin D, and (iv) metabolic conversion by hydroxylation reactions. The main metabolites produced by metabolic conversion (iv) are known to be: 25-hydroxyvitamin D produced by hydroxylating vitamin D at the 25-position by vitamin D-25-hydroxylase; 1 α, 25-dihydroxyvitamin D produced by hydroxylating the 1 α position of hydroxyvitamin D by 25-hydroxyvitamin D-1 α -hydroxylase; or 24, 25-dihydroxyvitamin D or 1 alpha, 24, 25-trihydroxyvitamin D produced by the 24-hydroxylase reducing the hydroxyl group at the 24-position of the metabolite. The vitamin D metabolism-regulating activity can be described as an activity of regulating an activity of an enzyme involved in producing these vitamin D metabolites, a change in gene expression or protein expression amount of the enzyme.

1. DNA encoding polypeptides that modulate phosphate metabolism, calcium metabolism, calcification and vitamin D metabolism

(1) Cloning of DNA

SEQ ID NO: the DNA shown in 1 is one of the DNAs of the present invention, which is obtained by preparing a cDNA library from a part of a tumor excised from a patient suspected of having tumor-induced osteomalacia and screening the library.

Tumor-induced osteomalacia is a disease associated with the presence of tumors, resulting in hypophosphatemia and osteomalacia due to insufficient calcification of bone tissue, characterized by the disappearance of the symptoms of resected tumors. Tumor extracts have been reported to promote urinary phosphate excretion in mice (Popvtzer, M.M. et al, Clinical Research 29: 418A, 1981), and also in an experiment in which excised tumors induced hypophosphatemia when transplanted into mice (Miyauchi, A. et al, J.Clin.Endocrinol.Metab.67: 46-53, 1988). Thus, unknown factors that may be tumor-produced and secrete a system have been considered.

We used one case associated with the tumor described by Fukumoto, S et al (Bone 25: 375-377, 1999). In this case, surgical removal of the tumor allowed significant recovery from hypophosphatemia. In addition, in this case, the size of the tumor is only 1 cm in diameter. Based on the inference that such small tissues are capable of producing and secreting an active agent that induces hypophosphatemia and systemic osteomalacia, we expect that the tumor-derived cDNA libraries we have prepared are more likely to contain at least a partial fragment of the gene encoding the active agent involved in the induction of these clinical conditions than cDNA libraries derived from other tissues. Accordingly, in order to identify the fragment sequence of the gene encoding a tumor-derived active substance, cDNA fragments having a particularly high content in a tumor cDNA library were extracted by differential screening.

Next, the nucleotide sequences of the resulting cDNA fragments were determined and compared with each other. Contigs were then prepared to group sequences that were each considered to be from the same gene based on the overlap of nucleotide sequences. The nucleotide sequence thus obtained was subjected to homology search with a nucleotide sequence registered in Genbank (a database provided by the national center for biotechnology information, hereinafter also referred to as NCBI). In this way, a nucleotide sequence particularly abundant in tumor cDNA libraries was obtained, i.e. SEQ ID NO: 1 from nucleotide 1522 to 2770. This sequence is identical to a portion of the human sequence 12p13BAC RPCI11-388F6, deposited under accession number AC008012 in Genbank. The registration sequence is considered to represent a partial sequence of the 12p13 region in the human chromosomal sequence. Although the position and nucleotide sequence information of the expected gene is shown in the registration sequence, SEQ ID NO: 1 from nucleotide 1522 to 2770, and the sequence of SEQ ID NO: 1 is not included in any indicated region of the intended gene.

Then according to SEQ ID NO: 1 from nucleotides 1522 to 2770, and isolating and identifying a contiguous nucleotide sequence contained in a tumor cDNA library, thereby obtaining the nucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 1 has an open reading frame (hereinafter also referred to as ORF) encoding a polypeptide consisting of SEQ ID NO: 2, which polypeptide is predicted to have a secretion signal. We believe this is a polypeptide with a novel sequence because the amino acid sequence of this polypeptide is not registered with the amino acid sequence database of Genbank. By using SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2, we have identified that the amino acid sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10, which are considered to be murine orthologs of said molecule. As described below, recombinant proteins prepared based on the amino acid sequence of human are active in mice. Thus, we convert SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 10 or the full-length mouse sequence (biochem. Biophys. Res. Commun.2000, 277(2), 494-498), the present invention allows easy assessment of proteins having biological activity identical or similar to the polypeptide of the present invention, in which an amino acid other than the conserved amino acids is substituted.

(2) Determination of nucleotide sequence

We determined the nucleotide sequence of the DNA described in (1). The nucleotide sequence can be determined by known techniques, such as the Maxam-Gilbert chemical modification or dideoxynucleotide chain termination method using M13 phage. Typically, sequencing is performed using an automated sequencer (e.g., 373A DNA sequencer produced by PERKIN-ELMER).

An example of the nucleotide sequence of the DNA of the present invention is SEQ ID NO: 1, an example of an amino acid sequence of a polypeptide of the invention is SEQ ID NO: 2. the amino acid sequence may contain a mutation such as deletion, substitution or addition of 1or several amino acids as long as the polypeptide consisting of the amino acid sequence has hypophosphatemia-inducing activity, phosphate transport-inhibiting activity, calcification-inhibiting activity or vitamin D metabolism-regulating activity.

For example, the peptide can be derived from SEQ ID NO: 2, preferably 1 to 10, more preferably 1 to 5 amino acids; SEQ ID NO: 2 by adding 1or several, preferably 1 to 10, more preferably 1 to 5 amino acids; alternatively, other amino acids may be substituted for SEQ ID NO: 2, preferably 1 to 10, more preferably 1 to 5 amino acids.

Furthermore, as a substitution method, conservative substitutions may be made within a family that maintains the properties of amino acids to some extent. Examples of families, which are generally classified according to the characteristics of amino acid side chains, are as follows.

(i) Family of acidic amino acids: aspartic acid, glutamic acid

(ii) Family of basic amino acids: lysine, arginine, histidine

(iii) Family of non-polar amino acids: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan

(iv) Family of uncharged polar amino acids: glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine

(v) Aliphatic hydroxy amino acids: serine, threonine

(vi) Family of amino acids containing an amido group: asparagine, glutamine

(vii) Aliphatic amino acids: alanine, valine, leucine, isoleucine

(viii) Aromatic amino acids: phenylalanine, tryptophan, tyrosine

(ix) Hydrophobic amino acids: leucine, isoleucine, valine

(x) Small amino acid family: alanine, serine, threonine, methionine, glycine

An example of substitution is represented by SEQ ID NO: 2 by replacing Arg at position 176 and/or Arg at position 179 with another amino acid, preferably Ala, Gln or Trp, such that the cleavage site is prevented or inhibited therein. Furthermore, the polypeptides of the invention also encompass polypeptides consisting of the amino acid sequence set forth in SEQ ID NO: 2 by deletion of 10 or more amino acids from the N-terminus, C-terminus or both termini (terminal deletion type). Examples of such terminal deletion forms include those represented by SEQ ID NO: 2 by deletion of 20, 40, 45 or 50 amino acids at the C-terminus and/or by deletion of 24 or 33 amino acids at the N-terminus. The embodiment of the terminal deletion type is as follows.

Number of amino acids deleted at N-terminus	Number of C-terminal deleted amino acids	In SEQ ID NO: 2 (nucleotide numbering in SEQ ID NO: 1)
Number of amino acids deleted at N-terminus	Number of C-terminal deleted amino acids	In SEQ ID NO: 2 (nucleotide numbering in SEQ ID NO: 1)	33 at N terminal	Not deleted	34-251(232-885)
33 at N terminal	20 at C terminal	34-231(232-825)	33 at N terminal	Not deleted	34-251(232-885)
33 at N terminal	20 at C terminal	34-231(232-825)	33 at N terminal	40 at C terminal	34-211(232-765)
33 at N terminal	The C terminal is 45	34-206(232-750)	33 at N terminal	40 at C terminal	34-211(232-765)
33 at N terminal	The C terminal is 45	34-206(232-750)	33 at N terminal	C terminal 50	34-201(232-735)
24 at N terminal	Not deleted	25-251(205-885) (corresponding to 1 to 681 in SEQ ID NO: 3 and SEQ ID NO: 4)	33 at N terminal	C terminal 50	34-201(232-735)
24 at N terminal	Not deleted		24 at N terminal	20 at C terminal	25-231(205-825)
24 at N terminal	40 at C terminal	25-211(205-765)	24 at N terminal	20 at C terminal	25-231(205-825)
24 at N terminal	40 at C terminal	25-211(205-765)	24 at N terminal	The C terminal is 45	25-206(205-750)
24 at N terminal	C terminal 50	25-201(205-735)	24 at N terminal	The C terminal is 45	25-206(205-750)
24 at N terminal	C terminal 50	25-201(205-735)	Not deleted	20 at C terminal	1-231(133-825)
Not deleted	40 at C terminal	1-211(133-765)	Not deleted	20 at C terminal	1-231(133-825)
Not deleted	40 at C terminal	1-211(133-765)	Not deleted	The C terminal is 45	1-206(133-750)
Not deleted	C terminal 50	1-201(133-735)	Not deleted	The C terminal is 45	1-206(133-750)

In addition to the above SEQ ID NO: 2, and the polypeptide of the present invention also encompasses mutant fragments obtained by deleting, substituting or adding 1or several amino acids from these terminal deletion-type polypeptides. In the above table SEQ ID NO: 2 represents the number in parentheses after the amino acid position in SEQ ID NO: 1, or a nucleotide position of the nucleotide sequence shown in figure 1. Thus, the present invention also encompasses DNAs consisting of the nucleotide sequences shown at these positions or DNAs which hybridize with these DNAs under stringent conditions.

In the present invention, in order to introduce a mutation into at least a part of the amino acid sequence of the polypeptide of the present invention, a technique of introducing a mutation into a nucleotide sequence of DNA encoding the amino acid is employed.

Mutations can be introduced into DNA by known techniques, such as the Kunkel method or the Gappeddulex method, or corresponding methods. For example, mutations are introduced based on a site-directed mutagenesis method using a mutation oligonucleotide as a primer. Furthermore, a kit for generating a mutation, such as Mutan-K (TAKARA), Mutan-G (TAKARA), LA PCR in vitro mutation kit (TAKARA), and the like, may also be used to introduce a mutation.

In addition, the DNA of the present invention also encompasses a DNA which hybridizes with a probe prepared from the above-mentioned DNA of the present invention (SEQ ID NO: 1, 3, 5, 7 and 9) under stringent conditions and encodes a polypeptide having a hypophosphatemia-inducing activity, a phosphate transport inhibitory activity, a calcification inhibitory activity or a vitamin D metabolism-regulating activity. The probes used herein have sequences identical to SEQ id no: 1, 3, 5, 7 or 9 or a sequence complementary to 17 or more contiguous nucleotides (partial sequence) thereof.

Here, the term "stringent conditions" satisfies the conditions that the salt concentration is 750mM or more, preferably 900mM or more, and the temperature is 40 ℃ or more, preferably 42 ℃. Specifically, stringent conditions as used herein include 6 XSSC, 5 XDenhardt, 0.5% SDS, 50% formaldehyde and 42 ℃. In addition, 6 XSSC represents 900mM NaCl and 90mM sodium citrate. Denhardt's solution (Denhardt) contains BSA (bovine serum albumin), polyvinylpyrrolidone and Ficoll 400. The 50x Denhardt solution consisted of 1% BSA, 1% polyvinylpyrrolidone and 1% Ficoll400(5x Denhardt represents one tenth of the 50x Denhardt concentration).

Once the nucleotide sequence of the DNA of the present invention has been determined, the DNA of the present invention can be obtained by chemical synthesis or by PCR from primers synthesized from the determined nucleotide sequence.

2. Recombinant vector containing the DNA of the invention and preparation of transformant

(1) Preparation of recombinant vectors

The recombinant vector of the present invention can be obtained by ligating the DNA of the present invention into an appropriate vector. The vector for inserting the DNA of the present invention is not particularly limited as long as it can replicate in a host. Examples of such vectors include plasmid DNA and phage DNA.

Examples of the plasmid DNA include plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC118 and pUC119), plasmids derived from Bacillus subtilis (e.g., pUB110 and pTP5), and plasmids derived from yeast (e.g., YEp13, YEp24 and YCp 50). An example of phage DNA is lambda phage. In addition, animal viral vectors such as retroviruses, adenoviruses or vaccinia viruses, or insect viral vectors such as baculoviruses may also be used. Alternatively, a fusion plasmid to which GST, His-tag, or the like is ligated can be used.

For inserting the DNA of the present invention into a vector, a method may be used which comprises first cleaving the purified DNA with an appropriate restriction enzyme and ligating the resulting cleaved DNA vector to a vector by inserting the cleaved DNA into a restriction enzyme site or a multiple cloning site of the DNA of an appropriate vector.

The DNA of the present invention must be integrated into a vector so that the DNA can perform its function. For the vector of the invention, cis-acting elements such as an enhancer, a splicing signal, a poly A addition signal, a selection marker and a ribosome binding sequence (SD sequence) may be linked if necessary in addition to the promoter and the DNA of the invention. In addition, examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, and a neomycin resistance gene.

(2) Preparation of transformant

The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host to enable expression of the target gene. The host used herein is not particularly limited as long as it can express the DNA of the present invention. Examples of such hosts include bacteria: escherichia, such as Escherichia coli; bacillus, such as bacillus subtilis; pseudomonas, such as Pseudomonas putida; or a yeast such as Saccharomyces cerevisiae or Schizosaccharomyces pombe. In addition, animal cells such as COS cells, CHO cells or HEK293 cells, and insect cells such as Sf9 or Sf21 can also be used.

When a cell such as Escherichia coli is used as a host, it is preferable that the recombinant vector of the present invention can autonomously replicate in bacteria, and comprises a promoter, a ribosome binding sequence, the DNA of the present invention, and a transcription termination sequence. In addition, a gene that regulates a promoter may be contained. Examples of Escherichia coli include JM109 and HB101, and an example of Bacillus subtilis is Bacillus subtilis. Any promoter can be used as long as it can be expressed in a host such as E.coli. For example, a promoter derived from Escherichia coli, such as trp promoter, lac promoter, PL promoter or PR promoter or T7 promoter derived from phage, etc. Artificially designed and modified promoters, such as the tac promoter, may also be used. The method employed herein for introducing the recombinant vector into a bacterium is not particularly limited as long as it is a method for introducing DNA into a bacterium. Examples of such methods include a method using calcium ions and an electroporation method.

When yeast is used as the host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris can be used. The promoter used in this case is not particularly limited as long as it can be expressed in yeast. Examples of such promoters include the gal1 promoter, gal10 promoter, tripsin promoter, MF α 1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and AOX1 promoter. The method for introducing the recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. Examples of such methods include electroporation, spheroplast and lithium acetate.

When animal cells are used as the host, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3 cells, human FL, HEK293, HeLa, Jurkat cells, and the like can be used. As the promoter, SR α promoter, SV40 promoter, LTR promoter, β -actin promoter, and the like can be used. In addition, an early gene promoter of human cytomegalovirus or the like can be used. Examples of introducing the recombinant vector into animal cells include electroporation, calcium phosphate method and lipofection.

When insect cells are used as the host, Sf9 cells, Sf21 cells, and the like can be used. As a method for introducing the recombinant vector into insect cells, a calcium phosphate method, a lipofection method, an electroporation method, and the like can be used.

3. Polypeptides having hypophosphatemia-inducing activity

There have been several attempts to isolate and identify tumor-derived factors with hypophosphatemia-inducing activity from tumor-induced osteomalacia. Thus, the polypeptides of the invention exhibit the properties of a novel secreted factor produced by the tumor of tumor-induced osteomalacia. The expected biological activity of the hypophosphatemia inducing factor is reported as follows.

Effect on promoting phosphate excretion into urine:

aschinberg, l.c. et al, j.pediatrics 91: 56-60, 1977, Lau, K, et al, Clinical Research 27: 421A, 1979, Miyauchi, a. et al, j.clin.endocrinol.meta.67: 46-53, 1988

Inhibition of phosphate transport activity in tubular epidermal cells:

cai, q, et al, n.engl.j.med.330: 1645-1649, 1994, Wilkins, G.E., et al, J.Clin.Endocrinol.Metab.80: 1628-: 159-169, 1996

Inhibition of 25-hydroxyvitamin D-1 α -hydroxylase activity:

miyauchi, a. et al, j.clin.endocrinol.metab.67: 46-53, 1988

In particular, an unknown molecule that directly inhibits phosphate reabsorption in the kidney has been proposed as Phosphatonin (Econs, M.J. & Drezner, M.K., Engl. J. Med 330: 1679-. It has also been suggested that an unknown molecule with such biological activity is also present in XLH. The clinical findings for patients with XLH are characterized by hypophosphatemia with enhanced urinary phosphate excretion, which is the same as in tumor-induced osteomalacia patients, and XLH progresses to osteomalacia or rickets due to insufficient calcification in the bone tissue. It has been demonstrated that the gene responsible for XLH is a gene encoding a endopeptidase-like protein, called PHEX. Recently, partial deletions of the PHEX-encoding gene were found in Hyp mice, a naturally mutated mouse known to express phenotypic characteristics similar to XLH, suggesting that Hyp mice are considered to be correct for the XLH model (Strom, T.M. et al Human molecular genetics 6: 165-171, 1997). The hypophosphatemia-inducing factor in Hyp mice is a humoral factor, which has been confirmed in xenobiotic symbiotic experiments using Hyp mice and normal mice (Meyer, R.A., et al, J.bone Miner.Res.4: 493-500, 1989). In this experiment, normal mice had decreased blood phosphate levels and increased urinary phosphate excretion. Therefore, it is considered that the humoral hypophosphatemia inducing factor present in Hyp mice acts on normal mice. The relationship between PHEX, which is expected to have peptide chain cleavage activity, and this unknown hypophosphatemia-inducing factor has not been clarified so far. However, some assumptions concerning the relationship between PHEX and the unknown hypophosphatemia-inducing factors have been made that PHEX may modulate the activity of the same factor as that found in XLH, and that this is also a possibility (Drezner, M.K. Kidney Int 57: 9-18, 2000). According to this hypothesis, both PHEX and the hypophosphatemia-inducing factor are conventionally expressed in the same cell, and PHEX acts inhibitively on the hypophosphatemia-inducing factor. This function of PHEX is reduced or eliminated in XLH patients, and thus the activity of hypophosphatemia-inducing factors is strongly expressed. It is believed that both PHEX and hypophosphatemia-inducing factors are elevated in tumor-induced osteomalacia, and finally the active hypophosphatemia-inducing factors are present in amounts exceeding normal levels. It is also believed that this hypophosphatemia-inducing factor acts inhibitively on the phosphate transport activity of NPT2, which is one of the phosphate carriers in the kidney. Many attempts have been made to find this unknown hypophosphatemia inducing factor, but none of them have identified this molecule. According to a study by Cai et al, the molecular weight of this hypophosphatemia inducing factor is expected to be in the range of 8kDa to 25kDa (Cai, Q. et al, N.Engl. J.Med.330: 1645-1649, 1994), but Rowe et al suggested that two proteins of 56kDa and 58kDa are candidate molecules. Recently, Rowe et al filed a patent application (WO99/60017) on a polypeptide comprising 430 amino acid residues, which is a tumor-derived phosphate metabolism modulator for tumor-induced osteomalacia. However, the polypeptide disclosed in this application is a partial sequence of a protein originally present, and biological activities associated with the hypophosphatemia-inducing activity are not disclosed. Recently, a polypeptide corresponding to the full-length molecule named MEPE disclosed in this patent has been reported, but its hypophosphatemia-inducing activity has not been disclosed at the same time (Rowe, P.S.N., et al, Genomics, 67: 54-68, 2000). In addition, there is no recognizable sequence or structural similarity between the molecule and the polypeptide of the invention.

As described above, it is inferred that a physiologically active factor having an activity of inducing hypophosphatemia exists, but the identity thereof has not been determined so far. In the present invention, we have ascertained the entity of the polypeptide, as well as the gene sequence encoding the polypeptide. Furthermore, as described later, we prepared the polypeptide of the present invention, suggesting that the product may be a regulator of phosphate metabolism, calcium metabolism and vitamin D metabolism or calcification and osteogenesis, and that the product may be a pharmaceutical composition. In addition, we have demonstrated that the antibodies of the invention can be used not only in therapy, but also in clinical testing and diagnosis. Furthermore, we show that DNA encoding the polypeptide of the present invention can be used for diagnosis of genetic diseases, and polymorphism diagnosis of phosphate metabolism, calcium metabolism and bone metabolism.

The polypeptide having hypophosphatemia-inducing activity of the present invention can be prepared by, for example, a method comprising the step of preparing a polypeptide comprising SEQ ID NO: 1 in a form capable of being expressed into a suitable host cell to prepare a transformant cell, and then allowing the DNA introduced into the transformant cell to express. In addition, the polypeptide chain prepared in this manner can be modified by a protein modification mechanism of the host, such as cleavage or addition of a sugar chain.

The polypeptide of the present invention can be obtained by culturing the above transformant and then collecting the cultured product. The term "culture product" means all cultured cells or cultured microorganisms, or lysed cells or microorganisms, except for the culture supernatant.

The transformant of the present invention can be cultured by any conventional host culture method.

As long as it contains a carbon source, a nitrogen source, inorganic salts, etc. which can be assimilated by the microorganism to efficiently culture the transformant, a natural or synthetic medium can be used for culturing the transformant obtained by using the microorganism such as Escherichia coli or yeast as a host. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose or starch, organic acids such as acetic acid or propionic acid, and alcohols such as ethanol or propanol. Examples of the nitrogen source include ammonia, ammonium salts of organic or inorganic acids such as ammonium chloride, ammonium sulfate, ammonium acetate or ammonium phosphate, or other nitrogen-containing compounds, as well as peptone, meat extract, and corn steep liquor. Examples of the minerals include potassium dihydrogenphosphate, dipotassium hydrogenphosphate, magnesium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, copper sulfate and calcium carbonate.

The culture is usually carried out under aerobic conditions, for example, shaking culture at 37 ℃ or aeration-stirring for 4 to 48 hours. During the cultivation, the pH is maintained between 6.0 and 8.0. The pH is adjusted with an inorganic or organic acid, an alkali solution, or the like. In the culture, if necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium.

When a microorganism transformed with an expression vector using an inducible promoter is cultured, an inducer may be added to the medium if necessary. For example, when a microorganism transformed with an expression vector containing a T7 promoter is cultured, the promoter can be induced with isopropyl-. beta. -D-thiogalactoside (IPTG), and IPTG or the like can be added to the medium. Further, when a microorganism transformed with an expression vector containing a trp promoter is cultured, the promoter can be induced with indoleacetic acid (IAA), and IAA or the like can be added to the medium.

Examples of a medium for culturing a transformant obtained by using an animal cell as a host include RPMI1640 medium, DMEM medium, or a medium supplemented with fetal bovine serum or the like, which are commonly used. The culture is usually carried out at 37 ℃ and 5% CO₂Under the condition of 1 to 10 days. In the culture, if necessary, an antibiotic such as kanamycin or penicillin may be added to the medium. After culturing, when the polypeptide of the present invention is produced in a microorganism or a cell, the target polypeptide can be collected by disrupting the microorganism or the cell by sonication, repeated freezing and thawing, homogenization treatment, or the like. When sending outWhen the polypeptide is produced outside the bacteria or cells, the bacteria or cells are removed by centrifugation or the like using the whole culture medium. Then, conventional biochemical methods for isolating and purifying proteins (such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography and affinity chromatography) may be used alone or in combination to isolate and purify the polypeptide of the present invention from the above-mentioned culture product.

As described above for XLH, PHEX, which is considered to be an endopeptidase, is important in regulating the induction factor of hypophosphatemia. Thus, the invention has the sequence of SEQ ID NO: 2, may have altered activity after further modification and cleavage. In the present invention, a clonal cell line capable of producing a recombinant polypeptide having 6 consecutive His at the C-terminus of a polypeptide of the present invention was prepared using CHO ras-clone 1 cells as a host. The cell line was deposited at national institute of advanced Industrial science and technology, International patent organism depositary (Chuo 6, 1-1-1, Higashi, Tsukuba-shi, Ibaraki, Japan) (accession number FERM BP-7273, initial deposit: 8/11/2000).

When detecting polypeptides of the present invention produced by cell lines and secreted into the culture medium, gene products of different sizes were probed by Western blotting using antibodies recognizing His-tag sequences, as shown in fig. 2. The protein corresponding to each band was isolated and the N-terminal amino acid sequence was determined. Thus, SEQ ID NO: 4 and SEQ ID NO: 8 are identical in N-terminal amino acid sequence. It is also contemplated that a protein having a preceding sequence may correspond to a protein from which a signal sequence has been removed, and a protein having a subsequent sequence may correspond to a protein which has been cleaved by an enzyme, such as an endopeptidase.

Currently, furin is one of the known proteolytic enzymes that recognizes RXXR. Indeed, when the polypeptides of the invention are expressed in furin deficient cell lines, no fragments can be detected. Moreover, when the recombinant protein α 1-PDX having furin inhibitory activity was co-expressed with the polypeptide of the present invention, the amount of cleaved product in the supernatant was significantly reduced.

Thus, the invention also encompasses a method of producing a polypeptide of the invention comprising the step of culturing a furin deficient cell, or in the presence of an agent which inhibits the activity of a furin enzyme.

Cai et al have proposed a phosphate transport inhibitory activity in the supernatant of a culture solution obtained by culturing tumor-derived cells of tumor-induced osteomalacia, showing a molecular weight in the range of 8kDa to 25kDa when measured by fractionation using a dialysis membrane. It is also conceivable to use the invention with the nucleic acid sequence of SEQ ID NO: 4 to the amino acid sequence set forth in SEQ ID NO: 2 between residue 179 Arg and residue 180Ser, may alter the activity.

The polypeptides of the invention have the ability to inhibit the phosphate transport activity of the kidney proximal tubule epidermal cells, a form of hypophosphatemia-inducing activity, as shown in table 2 (example 7). Most of the free inorganic phosphate in the blood is filtered in the glomeruli, with about 80% to 90% of the inorganic phosphate being reabsorbed in the proximal tubules of the kidney.

Resorption is via phosphate transport by the Na-dependent phosphate type II carrier present on the proximal luminal surface of the tubules. The polypeptides of the invention have the ability to inhibit phosphate transport activity. This means that the polypeptide of the present invention can promote the excretion of phosphate urine in vivo. Therefore, it is considered that the polypeptide of the present invention induces hypophosphatemia by exerting its activity of inhibiting phosphate reabsorption in the kidney, particularly phosphate transport in the proximal tubular kidney cells, and therefore the polypeptide of the present invention is expected to be the same substance as the above phosphatin.

Recently, Na-dependent phosphate carriers have been identified in the intestinal tract. This vector was named type IIb because it has very high homology to the type II Na-dependent phosphate vector present in the kidney. It is envisioned that the polypeptides of the invention are also capable of inhibiting the presence of type IIb Na-dependent phosphate carriers on the luminal surface of the intestinal tract, similar to type II Na-dependent phosphate carriers in the kidney. This may be considered as a form of hypophosphatemia-inducing activity.

The in vivo activity of the polypeptide of the present invention was evaluated by an experiment in which recombinant cells expressing the above polypeptide were subcutaneously transplanted into nude mice.

The transplanted cells in this experiment were grown in the subcutaneous space of nude mice and then allowed to form tumors. The polypeptides of the invention produced and secreted by the cells as the tumor is formed are characterized by the ability to be released into the body fluids of mice, thereby reproducing the release of tumor-derived humoral factors in tumor-induced osteomalacia in this animal model. In this model experiment, as shown in Table 4 (example 11), mice transplanted with cells expressing the polypeptide of the present invention developed marked hypophosphatemia compared with control mice that developed tumors by transplantation of CHO cells into which the DNA of the present invention was not introduced, or mice that did not develop tumors. In contrast, the polypeptide of the present invention exhibits hypophosphatemia-inducing activity. In addition, the experiment also showed that phosphate reabsorption was also decreased and phosphate reabsorption in the kidney was inhibited. Therefore, it was concluded that the polypeptide of the present invention is a hypophosphatemia-inducing factor of tumor-induced osteomalacia.

On the other hand, in the above model experiment, calcium deficiency was found in mice transplanted with recombinant cells capable of producing the polypeptide of the present invention. Therefore, it was suggested that the polypeptide of the present invention is also a calcium deficiency-inducing factor. CHO cells expressing the polypeptide of the present invention were transplanted into nude mice in the experiment described in example 16, which showed a continuous decrease in serum 1 alpha, 25-dihydroxyvitamin D levels. When the mutant-introduced polypeptide of the present invention or the wild-type full-length polypeptide of the present invention was administered to normal mice three times, serum 1 α, 25-dihydroxyvitamin D levels were reduced in both, as described in examples 19 and 20. In addition, as described in example 24, a decrease in serum 1 α, 25-dihydroxyvitamin D levels was observed within a few hours after a single administration of the polypeptide of the present invention. Thus, it is envisaged that activity leading to a reduction in 1 α, 25-dihydroxyvitamin D levels is a major biological or physiological effect of the polypeptides of the invention.

As described above, serum 1 α, 25-dihydroxyvitamin D levels are regulated by 1 α -hydroxylase and 24-hydroxylase. As described in example 16, the effect of the polypeptides of the invention on lowering serum 1 α, 25-dihydroxyvitamin D levels is accompanied by fluctuations in the expression of these metabolic enzymes. Furthermore, as described in example 24, 1 hour after administration of the polypeptide of the present invention, a decrease in the gene transcription product of 1. alpha. -hydroxylase responsible for the production of active vitamin D metabolites and an increase in the gene transcription product of 24-hydroxylase responsible for the decomposition of active vitamin D metabolites were observed. After these fluctuations in expression, serum 1 α, 25-dihydroxyvitamin D levels gradually decreased, suggesting that the effect of the polypeptides of the invention in decreasing 1 α, 25-dihydroxyvitamin D levels is due at least to the inhibition of 1 α -hydroxylase gene expression and the enhancement of 24-hydroxylase gene expression.

In contrast, in a long-term transplantation experiment (44 to 46 days after transplantation) in which CHO cells expressing the polypeptide of the present invention were transplanted into nude mice as described in example 11, the expression of 1. alpha. -hydroxylase gene was elevated. The serum PTH levels of the mice at this time were shown to be significantly elevated compared to the control group. Therefore, it can be presumed that the enhanced expression of the 1 α -hydroxylase gene is due to the effect of high levels of PTH. Interestingly, however, the expression of the 24-hydroxylase gene continues to rise even under conditions of high PTH levels, which may be understood as the inability of high PTH levels to interfere with the regulation of 24-hydroxylase gene expression by the polypeptides of the invention. As described in example 11, serum 1 α, 25-dihydroxyvitamin D3 levels were not increased, although there were mice that exhibited clinical findings of severe hypophosphatemia-like or rickets-like. This suggests that the polypeptide of the present invention has the effect of continuously enhancing the expression of the 24-hydroxylase gene.

Nykjaer et al have disclosed that 25-hydroxyvitamin D is reabsorbed in the proximal tubule of the kidney (Cell, 96: 507-. Although not described herein, no significant changes in serum 25-hydroxyvitamin D levels were found in one experiment in which CHO cells expressing the polypeptides of the invention were transplanted into nude mice. Furthermore, no polypeptide of the invention was identified to affect the partial excretion (calculated as urine level/serum level/GFR) of major electrolytes (such as sodium, potassium or chloride, major amino acids or glucose), which suggests the fact that the tubular reabsorption function is not disrupted (t.shimada et al, proc.natl.acad.sci., in print). Therefore, it was demonstrated that the polypeptides of the present invention reduce serum 1 α, 25-dihydroxyvitamin D levels not by inhibiting 25-hydroxyvitamin D reabsorption in the renal tubules, but by acting specifically on the 1 α, 25-dihydroxyvitamin D synthesis pathway.

Serum 1 α, 25-dihydroxyvitamin D levels are known to be significantly reduced in tumor-induced osteomalacia. Furthermore, serum 1 α, 25-dihydroxystaphin D levels were within the normal range or slightly below the lower limit of the normal range in hypophosphatemic vitamin D-resistant rickets (XLH) or Hyp (model mice displaying clinical symptoms of XLH), despite severe drops in serum phosphate levels. Elevated expression of the 24-hydroxylase gene in Hyp mice is also known. In these clinical conditions, which progress to hypophosphatemia, expression of the 1 α -hydroxylase gene is normally elevated as serum phosphate levels decrease, thereby increasing serum 1 α, 25-dihydroxyvitamin D levels. Therefore, we believe that failure of any regulatory system that results in the loss of this normal physiological response is at least one of the causes of these clinical symptoms. These phenomena are similar to the physiological responses observed in mice as described in examples 11, 19 or 20, strongly suggesting that the polypeptides of the invention are able to reduce serum 1 α, 25-dihydroxyvitamin D3 levels in the above clinical conditions.

The X-ray image shown in FIG. 5 clearly shows that the degree of calcification of the skeletal tissue of the mouse into which the recombinant cell expressing the polypeptide of the present invention was transplanted was significantly reduced as compared with the control group. Thoracic deformity and the like are also observed, suggesting that the polypeptide of the present invention has an influence on bone formation.

In other words, it is thought that the polypeptide of the present invention has an effect of inhibiting calcification of bone tissue, or promoting accumulation of calcium and phosphate from bone tissue. It is also conceivable that a significant decrease in blood phosphate and calcium levels leads to a secondary inhibition of calcification of bone tissue.

In Hyp mice, bone-derived cells are thought to produce a factor that inhibits phosphate transport activity in the proximal tubule of the kidney (Lajeunesse, D. et al, Kidney Int.50: 1531-1538, 1996). In addition, osteoblasts of Hyp mice have been reported to release calcification-suppressing factors (Xiao, z.s., am.j.physiol., E700-E708, 1998). As mentioned above, clinical symptoms such as hypophosphatemia and bone tissue under-calcification are very similar to each other in XLH and tumor-induced osteomalacia, and these clinical symptoms may be induced by a humoral factor. These facts suggest the possibility that the renal phosphate transport inhibitory activity and the skeletal calcification inhibitory activity reported in these studies on Hyp mice are caused by the same factor. Furthermore, it has been reported that osteoblasts of Hyp mice show abnormal osteogenic function even in the state where calcium and phosphate levels are in the normal range (Ecarot, B. et al, J.bone Miner. Res.7: 215-. The polypeptide of the present invention has activity similar to that of the above-mentioned prospective factor of Hyp mice. Thus, it is contemplated that the polypeptides of the invention, in addition to their hypophosphatemia-inducing activity, have the effect of directly modulating calcification of bone tissue without mediation by calcium or phosphate metabolism.

In carrying out the present invention, dental matrix protein-1 (DMP-1) as shown in Table 1 in example 3 was obtained in addition to the gene encoding the polypeptide of the present invention. The gene is expressed abundantly in dentin, and the encoded protein is an extracellular matrix protein of dentin, which is thought to play an important role in the formation of calcified matrix of dentin. Similarly, the gene OST190 encoding matrix extracellular phosphorylated protein (MEPE) was obtained. The specific function of the molecule is unclear. Similarly, a gene encoding osteopontin was also obtained. MEPE, DMP-1 and osteopontin have common characteristics in that they are phosphorylated proteins containing RGD motif sequence, are rich in serine and threonine which can be phosphorylated, have high contents of acidic amino acids glutamic acid and aspartic acid, and show strong acidic protein characteristics. A characteristic acidic region, named ASARM sequence, is conserved in MEPE and DMP-1 (Rowe, P.S.N., et al, Genomics 67: 54-68, 2000), suggesting similarity in their physiological or functional significance. The interaction with inorganic calcium and/or phosphate at the onset of calcification is considered to be one of the functions of such characteristic proteins. In addition to osteoblasts and osteoclasts, expression of osteopontin genes in various cells, such as macrophages, has been reported. On the other hand, the expression of DMP-1 in skeletal tissues, particularly in bone cells, has been recently reported. Gene expression of MEPE in bone marrow tissue or osteosarcoma cells such as SaOS-2 is known. In the course of the present invention, the fact that acidic matrix proteins which are frequently found in calcified tissues are found together with the polypeptides of the present invention represents one aspect of the action of the polypeptides of the present invention. In particular, there is the possibility that the polypeptide of the invention induces the expression of a calcified tissue specific molecule represented by the above mentioned molecules, whereby said polypeptide regulates calcification, calcium metabolism and phosphate metabolism in a synergistic manner or the induced molecule regulates calcification, calcium metabolism and phosphate metabolism secondarily. It is also envisaged that the polypeptides of the invention may modulate bone metabolism by acting directly on osteoblasts, osteocytes and osteoclasts. Therefore, the polypeptide of the present invention can be used for treating metabolic bone diseases such as osteoporosis.

Recently, cells with osteoblast-like phenotype were shown to present sites of abnormal calcification, suggesting that calcification may occur through a similar mechanism to the process of calcification in bone tissue. Thus, it is envisioned that the polypeptides of the invention are effective for treating abnormal calcification by inhibiting the presence or function of these cells responsible for calcification.

In the present invention, the above-mentioned polypeptide may be modified. For example, polyethylene glycol, dextran, poly (N-vinyl-pyrrolidone), polypropylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylene polyol, polyvinyl alcohol, and the like can be suitably selected and used. Any known technique can be employed as a modification method. For example, a technique disclosed in detail in JP patent publication (PCT translation) No. 10-510980.

4. Antibodies against the polypeptides of the invention

The antibody of the present invention specifically reacts with the polypeptide of the present invention as described above. In the present invention, the term "antibody" means an entire antibody molecule or a fragment thereof (e.g., Fab or F (ab')₂Fragment) which may be a polyclonal or monoclonal antibody.

The antibody of the present invention can be prepared according to a conventional method. For example, antibodies can be prepared by in vivo methods, which involve immunizing an animal one or several times with antigen and adjuvant at weekly intervals (booster immunization), or in vitro methods, which involve isolating immune cells and sensitizing the immune cells using a suitable culture system. Examples of immune cells capable of producing the antibodies of the invention include spleen cells, tonsil cells and lymphocytes.

The polypeptide used as an antigen need not be the entire polypeptide of the present invention described above. A part of the polypeptide may be used as an antigen. When short peptides are used as the antigen, in particular, peptides as short as about 20 amino acid residues are used, such peptides being conjugated to a highly antigenic carrier protein such as keyhole limpet hemocyanin or bovine serum albumin by chemical modification or the like, or covalently conjugated to a branched backbone peptide other than the carrier protein such as lysine core MAP peptide (Posnett et al, J.biol.Chem.263, 1719-.

As the adjuvant, for example, Freund's complete or incomplete adjuvant, aluminum hydroxide gel, etc. can be used. For the animal immunized with the antigen, for example, a mouse, a rat, a rabbit, a sheep, a goat, a chicken, a cow, a horse, a guinea pig and a hamster can be used.

Polyclonal antibodies can be obtained by collecting blood from these immunized animals, separating the serum and purifying the immunoglobulin by one of the methods of ammonium sulfate precipitation, anion exchange chromatography and protein A or G chromatography or a suitable combination thereof. When the animal is a chicken, the antibody may be purified from chicken eggs.

Monoclonal antibodies are prepared by purifying culture supernatants of hybridomas prepared by fusing immune cells, which have been sensitized in vitro or taken from the above-mentioned animals, with parent cells capable of being cultured, or ascites from animals inoculated intraperitoneally with hybridomas. Common mature cell lines of animals, such as mice, can be used as parent cells. The cell line preferably used herein is drug-selective and has the property of being unable to survive in HAT selection medium (containing hypoxanthine, aminopterin and thymidine) without fusion, but being able to survive in a state of fusion with antibody-producing cells. Examples of such cell lines include X63, NS-1, P3U1, X63.653, SP2/0, Y3, SKO-007, GM1500, UC729-6, HM2.0 and NP4-1 cells.

Specific techniques for preparing monoclonal antibodies are as follows.

The polypeptide or a fragment thereof prepared as described above is administered as an antigen to the above-mentioned animal. The antigen dose per animal when adjuvant is used is 1 to 100 ug. Immunization is primarily carried out by intravenous, subcutaneous or intraperitoneal injection. The immunization interval is not particularly limited. The immunization is carried out 1 to 10 times, preferably 2 to 5 times, at intervals of several days to several weeks, preferably 1 to 3 weeks. After 1 to 10 days, preferably 1 to 4 days after the last immunization, antibody-producing cells are collected.

To obtain hybridomas, antibody-producing cells and primary cells (melanoma cells) are fused. Cell fusion was performed in serum-free medium (such as DMEM or RPMI-1640 medium) for culturing animal cells by mixing 5X10⁶To 1x10⁸Cells/ml antibody producing cells with 1X10⁶To 2x10⁷Cells/ml melanoma cells are mixed (preferably the ratio of antibody producing cells to melanoma cells is 5: 1) and the fusion reaction is carried out in the presence of a cell fusion promoting agent. Polyethylene glycol or the like having an average molecular weight of 1000 to 6000 daltons may be used as the fusion promoter. In addition, cell fusion devices (e.g., electroporation) available on the market can be utilizedElectrical stimulation fuses antibody-producing cells and melanoma cells.

After completion of cell fusion, target hybridomas are selected from the cells. The cell suspension is suitably diluted, for example, in RPMI-1640 medium containing fetal bovine serum at approximately 5X10⁵The concentration of cells/well was seeded into the microplate. Selection medium was added to each well and then cultured while changing the selection medium at the appropriate time. Finally, the cells proliferated after about 14 days of culture in the selection medium can be used as hybridomas. The culture supernatants of the proliferated hybridomas are screened for antibodies reactive with the polypeptide of the present invention. Screening of hybridomas can be carried out according to a conventional method, and the screening method is not particularly limited. For example, a part of the culture supernatant contained in a well in which a hybridoma grows is collected and then screened by an enzyme immunoassay, a radioimmunoassay, or the like.

Alternatively, monoclonal antibodies can be prepared by culturing immortalized antibody-producing cells obtained by infecting immune cells sensitized in vitro or derived from the above-mentioned immunized animal with a suitable virus (such as EB virus).

In addition to these cell engineering techniques, the monoclonal antibody can also be obtained by genetic engineering techniques. Such antibody genes can be amplified, for example, by PCR (polymerase chain reaction) from immune cells of the animal sensitized in vitro or as described above. The gene is introduced into a microorganism (e.g., Escherichia coli) to produce an antibody, or a phage expresses the antibody as a fusion protein on the cell surface.

The in vivo quantification of the polypeptide of the present invention using the antibody of the present invention enables elucidation of the relationship between the polypeptide of the present invention and clinical symptoms of various diseases. Furthermore, the antibodies can be used diagnostically or therapeutically and can be used for efficient affinity purification of the polypeptides of the invention.

It is estimated that some diseases are caused by an excessive action of the polypeptides of the invention resulting in a decrease in serum 1 α, 25-dihydroxyvitamin D levels. For example, although hypophosphatemic vitamin D resistant rickets (XLH) can progress to severe hypophosphatemia, no elevation in serum 1 α, 25-dihydroxyvitamin D3 levels was found. The cause of this is considered to be abnormality of the vitamin D metabolizing enzyme gene. In this disease, excessive action of the polypeptides of the invention may be involved. In Hyp (mouse model of XLH), enhanced expression of the 24-hydroxylase gene has been reported. This is consistent with the effect of the polypeptides of the invention in inducing enhanced expression of the 24-hydroxylase gene. Thus, it is expected that correction of serum levels of 1 α, 25-dihydroxyvitamin D3 may be used to treat this condition by administering antibodies against the polypeptides of the invention to normalize vitamin D metabolism. One disease that exhibits similar clinical manifestations as XLH is Autosomal Dominant Hypophosphatemic Rickets (ADHR). When cloning the polypeptide of the present invention, we conclude that the gene encoding the polypeptide is the gene responsible for ADHR, based on its chromosomal location. Recently, a gene causing ADHR has been analyzed, and the disease has been reported to be caused by a missense mutation in the gene encoding the polypeptide. We further demonstrated that this mutation confers enzyme resistance to this gene and suggested that the excessive action of this molecule is responsible for the disease. It is envisaged that antibodies directed against the polypeptides of the invention will be effective in the treatment of such diseases through their inhibitory effect. ADHR develops osteomalacia, mineral metabolism disorders and vitamin D metabolism disorders. Thus, in metabolic bone diseases closely related to these metabolic pathways, there may be some caused by the polypeptides of the present invention. It is expected that antibodies against the polypeptides of the invention will be effective against such diseases. It is known that inhibition of differentiation into adipocytes is one of the effects of 1 α, 25-dihydroxyvitamin D. And it is known that adipocytes in bone marrow increase with age. In this case, differentiation into adipocytes from hematopoietic cells, which are common in bone marrow and support hematopoiesis of bone marrow, precursor cells of adipocytes and stromal cells is enhanced. It is envisaged that in this process, the polypeptides of the invention may act excessively resulting in a decrease in 1 α, 25-dihydroxyvitamin D levels. Therefore, it is expected that the use of antibodies against the polypeptides of the present invention will increase blood or local 1 α, 25-dihydroxyvitamin D levels, inhibit differentiation into adipocytes, and increase decreased bone formation or hematopoietic potential. In addition, the antibody is expected to be effective in preventing obesity. It is envisaged that there are other diseases in which the polypeptides of the invention are involved. Such diseases can be screened by combining the immuno-quantitative method represented by ELISA with an antibody against the polypeptide of the present invention. Accordingly, the physiologically normal range of the polypeptide of the invention can be determined, and diseases deviating from this range can be identified. It is expected that antibodies against the polypeptide of the present invention may be useful for treating diseases in which the polypeptide of the present invention exhibits abnormally high blood levels as measured by the above method.

5. Pharmaceutical composition

(1) Pharmaceutical composition comprising the polypeptide of the invention

The polypeptides of the invention can be used as pharmaceutical compositions for diseases in which blood phosphate levels are undesirably elevated. In chronic renal failure, decreased excretion of phosphate from the kidneys results in increased blood phosphate levels. Hyperphosphatemia further aggravates kidney function and promotes secretion of parathyroid hormone from the parathyroid gland, thereby inducing secondary hyperparathyroidism. This disease results in itching of the skin and a decrease in intestinal calcium absorption caused by the disturbance of 1 α, 25-dihydroxyvitamin D3 synthesis in the kidney. In addition, the condition of excessive secretion of parathyroid hormone by blood phosphate retention promotes the loss of calcium from skeletal tissue. When this condition persists, fibroosteitis or parathyroid hyperplasia, a clinical symptom of renal osteodystrophy, occurs. The preferred method of escaping this condition is to increase the hypophosphatemia referred to above, but current medical approaches are not effective in controlling hypophosphatemia. In the stage where chronic renal failure can maintain the function of urination, the polypeptide of the present invention has the effect of correcting the blood phosphate level (phosphate transport inhibitory activity) by inhibiting type II Na-dependent phosphate carriers present in the epidermal cells of the proximal tubule of the kidney, thereby promoting the excretion of phosphate into the urine. In addition, the polypeptides of the invention are able to act on the intestinal tract to correct blood phosphate levels by inhibiting type II Na-dependent phosphate carriers in a similar manner as in the kidney, thereby reducing phosphate absorption into the body.

The polypeptide of the present invention can also be used as a pharmaceutical composition for diseases caused by abnormal calcium metabolism and phosphate metabolism. The term "abnormal calcium metabolism" refers to a state in which serum calcium levels deviate from a clinically defined normal range, or in which serum calcium levels are within a normal range, but the functions of the kidney, intestine, skeletal tissue and parathyroid gland are abnormally enhanced or decreased to maintain serum calcium levels, or in which serum calcium-regulating hormones such as parathyroid hormone, 1 α, 25-dihydroxyvitamin D3 or calcitonin exhibit abnormal values. Furthermore, the term "abnormal phosphate metabolism" refers to a state in which serum phosphate levels deviate from a clinically defined normal range, or serum phosphate levels fall within a normal range, but the function of phosphate balance in kidney, intestinal tract and skeletal tissues is abnormally enhanced or decreased.

Renal osteodystrophy and the secondary hyperparathyroidism described above presents a variety of clinical forms, such as dynamic deficiency skeletal disease, fibrous osteitis or their mixed phenotypes. In order to combat secondary hyperparathyroidism, parathyroid hormone is usually inhibited with 1 α, 25-dihydroxyvitamin D3, 1 α -hydroxyvitamin D3, and the like. When the parathyroid hormone values are not effectively inhibited, pulse therapy (hereinafter also referred to as "vitamin D pulse therapy") may be employed which involves administering an excess of 1 α, 25-dihydroxyvitamin D3 or 1 α -hydroxyvitamin D3. Normal serum parathyroid hormone levels are 65pg/ml or less. When parathyroid hormone levels are normal in this condition, it leads to a power-deficient skeletal disease, a form of renal osteodystrophy. Furthermore, when parathyroid hormone levels are elevated, fibroosteitis occurs, which is the opposite of the clinical symptoms described above. Recent guidelines for such diseases suggest maintaining parathyroid hormone levels at about 130 to 260 pg/ml. However, the root cause of abnormal metabolism is still unclear. Parathyroid hormone is known to be induced by elevated serum phosphate levels and inhibited by elevated serum calcium levels. Since the polypeptide of the present invention lowers blood phosphate levels and blood calcium levels, it is presumed that the polypeptide can alter the function of parathyroid hormone. In addition, it has been reported that 1 α, 25-dihydroxyvitamin D3 falls to the limit of measurement or lower in tumor-induced osteomalacia patients. It is therefore also concluded that the polypeptides of the invention may be involved in modulating the activity of 1 α, 25-dihydroxyvitamin D3. It is thought that the polypeptide of the present invention can be used as a clinically useful pharmaceutical composition for dynamic deficiency skeletal diseases or fibrous osteitis in renal osteodystrophy accompanied by abnormal regulation or impaired activity of the above-mentioned parathyroid hormone. Therefore, it is contemplated that administration of the polypeptides of the present invention provides a useful therapy for fibroosteitis or power-deficient bone disease in renal osteodystrophy, which are the opposite.

In addition, the polypeptide of the present invention may be used as medicine composition for treating abnormal calcification. Calcification of tissue other than bone tissue can impair biological function. In particular, functional disorders resulting from calcification of the heart or blood vessels can be life-threatening. One risk factor for such abnormal calcification is an increase in blood calcium and phosphate ion levels (hereinafter also referred to as "calcium-phosphate production"). In the above-mentioned secondary hyperparathyroidism treatment, when the clinical symptoms of hypophosphatemia are counteracted by vitamin D pulse therapy, calcium-phosphate production rises leading to abnormal calcification. Calcification of the cardiovascular system in hemodialysis patients is a serious problem. As shown in Table 4, the polypeptide of the present invention has an activity of significantly reducing serum calcium levels and phosphate levels, and thus it is expected to be effective against abnormal calcification and various diseases.

In addition, the polypeptide of the present invention can be used as a pharmaceutical composition against metabolic bone diseases. The polypeptides of the invention have strong regulatory activity on calcium metabolism, phosphate metabolism, calcification or vitamin D metabolism. Examples of hormones involved in calcium metabolism and phosphate metabolism include calcitonin, parathyroid hormone and 1 α, 25-dihydroxyvitamin D3. Calcitonin has serum calcium-lowering activity, while parathyroid hormone and 1 α, 25-dihydroxyvitamin D3 have serum calcium-raising activity. Parathyroid hormone has been reported to have an effect on phosphate excretion into urine, and calcitonin has similar activity. 1 alpha, 25-dihydroxyvitamin D3 has the activity of promoting the absorption of phosphate in the intestinal tract. As described above, each hormone has different activities for maintaining the balance between serum calcium and phosphate levels, but calcitonin and 1 α, 25-dihydroxyvitamin D are used as therapeutic agents for osteoporosis. In addition, parathyroid hormone is being developed as a therapeutic drug for osteoporosis.

Bone metabolism is characterized by the catabolism and anabolism of bone tissue, i.e., bone resorption and bone formation, which are combined. Continued administration of parathyroid hormone results in osteopenia. However, parathyroid hormone is known to promote bone formation when acted upon intermittently. Because the polypeptide of the present invention has the effect of regulating calcium metabolism and phosphate metabolism, it is expected that it is effective against metabolic bone diseases including osteoporosis when the polypeptide is used by selecting an appropriate method.

In addition, the polypeptide of the present invention can be used as a pharmaceutical composition for a disease or clinical condition in which serum 1 α, 25-dihydroxyvitamin D levels are undesirably elevated, or for a clinical condition accompanied by an adverse physiological response induced by serum 1 α, 25-dihydroxyvitamin D.

It is known that 1 α, 25-dihydroxyvitamin D acts on parathyroid glands to inhibit secretion of parathyroid hormone (PTH). Thus, clinically mature therapies for secondary hyperparathyroidism in chronic renal failure, particularly in cases with high serum PTH levels, involve intermittent administration of high concentrations of 1 α, 25-dihydroxyvitamin D. One drawback of this therapy is that it is prone to abnormal calcification. In patients with chronic renal failure with high serum phosphate levels, tissue calcification other than skeletal tissue, caused by administration of 1 α, 25-dihydroxyvitamin D, is often observed. Since the polypeptide of the present invention has the effect of promoting the rapid decrease of serum 1 α, 25-dihydroxyvitamin D within several hours after the administration of the polypeptide, the polypeptide can be used for the treatment and prevention of abnormal calcification caused by an excessively high level of 1 α, 25-dihydroxyvitamin D.

In addition, intermittent administration of high concentrations of 1 α, 25-dihydroxyvitamin D excessively inhibits PTH secretion, and therefore it can induce a power-deficient bone disease, progressing to clinical symptoms such as cessation of bone metabolism. In this case, it is expected that administration of the polypeptide of the present invention will result in a decrease in 1 α, 25-dihydroxyvitamin D in serum, promoting normal PTH secretion from parathyroid glands and recovery from dynamic deficient skeletal disease.

For vascular calcification, 1 α, 25-dihydroxyvitamin D has been reported as one of the calcification-promoting factors. The polypeptides of the invention can be used for treating or preventing clinical symptoms related to vascular calcification, such as arteriosclerosis caused by aging, diabetic angiopathy or calcification of the cardiovascular system of dialysis patients.

1 α, 25-dihydroxyvitamin D in serum is known to promote calcium absorption in the intestinal tract. The polypeptides of the invention may be used to correct hypophosphatemia by lowering serum 1 α, 25-dihydroxyvitamin D levels. The causes of hypercalcemia include excessive PTH due to primary hyperparathyroidism, excessive 1 α, 25-dihydroxyvitamin D levels associated with chronic granulomas, such as sarcoidosis or tuberculosis, or accelerated bone resorption by PTHrP produced in malignancies. In hypercalcemia, which is caused mainly by excess 1 α, 25-dihydroxyvitamin D and excess PTH or PTHrP, it is expected that administration of the polypeptide of the present invention will lower serum 1 α, 25-dihydroxyvitamin D levels, thereby alleviating hypercalcemia. In particular, macrophages that are activated in chronic granulomas (such as sarcoidosis or tuberculosis) have 1 α -hydroxylase activity and overproduce 1 α, 25-dihydroxyvitamin D3. It is expected that the polypeptide of the present invention may directly decrease the 1 α -hydroxylase activity.

Given that 1 α, 25-dihydroxyvitamin D promotes osteoclast differentiation, administration of the polypeptide of the present invention is expected to inhibit bone resorption. In vitro, 1 α, 25-dihydroxyvitamin D is known to be a strong osteoclast differentiation-inducing factor. Excessive bone resorption by osteoclasts leads to osteopenia, represented by osteoporosis. In such diseases where increased bone resorption is observed, the polypeptides of the invention are expected to restore normal bone circulation by temporarily reducing serum 1 α, 25-dihydroxyvitamin D levels. In addition to inhibiting osteoclast differentiation in vitro, 1 α, 25-dihydroxyvitamin D is also considered to be a factor inhibiting bone formation in vivo in one bone transplantation experiment using a vitamin D receptor deficient mouse. From these viewpoints, it is expected that the polypeptide of the present invention is effective against osteopenic metabolic bone diseases because it can lower 1 α, 25-dihydroxyvitamin D. In addition, there is a report that 24, 25-dihydroxyvitamin D, which is one of the metabolites of vitamin D3, is administered to cause bone mass increase. The 24, 25-dihydroxyvitamin D is a product obtained by hydroxylating 25-hydroxyvitamin D with 24-hydroxylase. Since the polypeptide of the present invention has an effect of significantly enhancing the expression of 24-hydroxylase gene, it is expected that administration of the polypeptide will result in an increase in the level of 24, 25-dihydroxyvitamin D in blood and an increase in bone mass in the clinical state of bone diseases such as osteoporosis or bone dysplasia.

PTH is known to have a strong bone resorption promoting effect. However, by intermittent administration of PTH, bone turnover can be stimulated, and finally, an effect of increasing bone mass can be achieved. The physiological or biological activity of the polypeptide of the invention comprises: modulating the effect of 1 α, 25-dihydroxyvitamin D; effects on modulating serum calcium and serum phosphate levels; and modulating the effect of calcification. Therefore, it is thought that the effective action of the polypeptide on bone tissue can regulate bone turnover. Therefore, the polypeptide is expected to be effective against postmenopausal osteoporosis with enhanced bone turnover and senile osteoporosis with decreased bone turnover. There may be other diseases in which the polypeptide of the present invention is involved. Such diseases can be screened by an immunochemical detection method typified by ELISA in combination with the use of 1or more antibodies against the polypeptide of the present invention.

Accordingly, a physiologically normal range of the polypeptide of the present invention can be set, and diseases in which the level of the protein deviates from this range can be confirmed. It is expected that the polypeptide of the present invention can be used for the treatment of diseases in which the polypeptide shows abnormally low blood levels as measured by the above method.

(2) Pharmaceutical composition containing antibody of the invention

The antibody of the invention can be used as a pharmaceutical composition for resisting vitamin D rickets and tumor-induced osteomalacia. Neutralizing antibodies to the polypeptide may be obtained in polyclonal or monoclonal form by the above-mentioned methods of producing antibodies. A more suitable method for using the antibody as a pharmaceutical can be to prepare a human type antibody or a humanized antibody. The hypophosphatemia and osteomalacia of tumor-induced osteomalacia can be treated or improved by inhibiting the excessive activity of the polypeptide of the present invention. Administration of neutralizing antibodies against the polypeptides of the invention is expected to improve tumor-induced osteomalacia. In addition, since the hypophosphatemia-inducing factor and calcification-suppressing factor of XLH are considered to be equivalent to the polypeptide of the present invention, the neutralizing antibody can also be used as a therapeutic agent against vitamin D rickets including XLH.

The antibodies of the invention may be used as pharmaceutical compositions for diseases of abnormal calcium or phosphate metabolism, or metabolic bone diseases associated with the presence of an excess of the polypeptide of the invention. As described above, in chronic renal failure or hemodialysis patients, abnormalities in the mechanism for maintaining the steady state of calcium metabolism or phosphate metabolism occur, which may be caused by excessive production or accumulation of the polypeptide of the present invention. The polypeptides of the invention are known to regulate bone metabolism. It is therefore foreseen that metabolic bone diseases may also exist due to the excessive presence of the polypeptides of the invention. In this case, it is expected that these clinical symptoms can be treated or improved with an antibody against the polypeptide of the present invention.

Examples of diseases in which the antibody of the present invention can be used include at least one bone disease such as osteoporosis, vitamin D-resistant rickets, renal osteodystrophy, dialysis-related bone disease, hypercalcemia bone disease, paget's disease, and tumor-induced osteomalacia. Here, the bone disease may be a single disease, or a complication, or a bone disease complicated with a disease other than the above-mentioned diseases.

(3) Dosing regimens

The pharmaceutical composition containing the polypeptide of the present invention or the antibody thereof as an active ingredient may contain pharmaceutically acceptable carriers and additives. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, carboxymethyl sodium starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose and surfactants that can be used as pharmaceutically acceptable additives. The additives used herein are selected from the above either alone or in combination, depending on the dosage form employed in the present invention.

When the polypeptide or antibody of the present invention is used as a prophylactic or therapeutic agent for bone diseases, the subject to which the polypeptide or antibody is to be used is not particularly limited.

The polypeptides of the invention can be used as pharmaceutical compositions for modulating calcium metabolism, phosphate metabolism, calcification or vitamin D metabolism in biological tissues.

The antibodies of the invention as described above may be particularly useful for the treatment or prevention of at least one bone disease such as osteoporosis, vitamin D resistant rickets, renal osteodystrophy, dialysis related bone disease, bone disease with hypocalcemia, paget's disease, tumor induced osteomalacia. The bone disease to which the polypeptide or antibody of the present invention can be applied may be a disease, a complication thereof, or a bone disease complicated with a disease other than these diseases.

The prophylactic or therapeutic agent comprising the polypeptide or antibody of the present invention can be administered orally or parenterally for the polypeptide, and parenterally for the antibody.

When the polypeptide of the present invention is orally administered, the dosage form to be employed may be a solid preparation such as a tablet, granule, powder or pill, or a liquid preparation such as a liquid medicine or syrup, etc. In particular, granules and powders may be prepared in unit dosage form, i.e., capsules. For liquid formulations, dry products can be formulated for reconstitution for use.

In these dosage forms, solid preparations for oral administration usually contain, in their composition additives, usually pharmaceutically employed, such as binders, excipients, lubricants, disintegrants or wetting agents. In addition, liquid preparations for oral administration usually contain, in their composition additives, those usually employed in medicine, such as stabilizers, buffers, flavors, preservatives, flavors or coloring agents.

When the polypeptide or antibody of the present invention is administered parenterally, it may be formulated into injections, suppositories, and the like.

For injections, they are usually presented in unit dose ampoules, in one or more dose containers, or may be in powder form for reconstitution in a suitable carrier, e.g. sterile water, without pyrogens, for use. These dosage forms usually contain, in their composition additives, such as emulsifying or suspending agents, which are often employed in medicine. Examples of injection procedures include intravenous drip, intravenous injection, intramuscular injection, peritoneal injection, subcutaneous injection or intradermal injection. In addition, the dosage difference may vary greatly depending on the age of the subject to be used, the route of administration and the frequency of administration.

In this case, an effective amount to be administered is a combination of an effective amount of the polypeptide or antibody of the present invention and a suitable diluent and a pharmaceutically acceptable carrier, and in the case of the polypeptide, 0.01 to 100. mu.g, preferably 0.5 to 20. mu.g per dose per kg body weight. Furthermore, for the antibody, the effective dose is 0.1. mu.g to 2mg, preferably 1 to 500. mu.g/time/kg body weight.

6. Disease diagnostic agent

(1) The antibody or polypeptide of the present invention

The antibody of the present invention can be used for detecting or quantifying the polypeptide or metabolite of the present invention present in blood or urine, thereby elucidating the relationship between the polypeptide of the present invention and clinical symptoms, and can be used as a diagnostic agent for a related disease.

The term "associated disease" means a bone disease or a disease that develops at least one of the following abnormalities: abnormal calcium metabolism, abnormal phosphate metabolism, abnormal calcification and abnormal vitamin D metabolism. Examples of such diseases include: osteoporosis, vitamin D resistant rickets, renal osteodystrophy, dialysis related bone disease, bone disease with hypocalcemia, paget's disease, renal failure, renal phosphate leakage, tubular acidosis and fanconi syndrome.

Methods for quantifying the amount of binding molecules using antibodies are conventional, such as radioimmunoassay or enzyme immunoassay. The level of the polypeptide of the present invention in blood or urine determined by these methods may guide a new clinical diagnosis. For example, when a rickets patient shows a higher level of the polypeptide of the present invention in his blood than in a normal subject, it is likely that XLH or ADHR is present. In addition, according to the change in blood level of the polypeptide of the present invention, prognosis for progression to secondary hyperparathyroidism can be made for patients with chronic renal failure.

For tumor-induced osteomalacia, it is often difficult to find tumors. However, useful diagnostic measures can be established using the antibodies of the invention. For example, when a patient without a family history of rickets or osteomalacia shows significantly higher levels of the polypeptide of the invention in blood than normal individuals, it may be suspected of having tumor-induced osteomalacia.

(2) DNA of the present invention

In the present invention, detection of the abnormal DNA of the present invention in a patient having abnormal phosphate metabolism or calcium metabolism or metabolic bone disease can be used for diagnosis and prevention of disease.

The nucleotide sequence of the DNA of the present invention was searched in the Genbank nucleotide sequence database to show that the nucleotide sequence (three fragments) coincided with the human 12p13BAC RPCI11-388F6 (accession number AC008012) sequence. This fragmentation indicates that the nucleotide sequence of the DNA of the present invention is a cleavage product of the chromosomal sequence. It is thus apparent that the DNA encoding the polypeptide of the present invention comprises SEQ ID NO: 11, or a partial fragment thereof, of at least between nucleotides 498 and 12966 of the sequence set forth in seq id No. 11. Substitutions, insertions or deletions of nucleotides within this range will result in an enhancement, reduction or abolition of the biological and physiological activity of the polypeptides of the invention. The polypeptides of the present invention have a strong influence on phosphate metabolism, calcium metabolism, bone metabolism and vitamin D metabolism. Therefore, when SEQ ID NO: 11or a partial region thereof (e.g., a sequence between nucleotides 498 and 12966) shows gene polymorphism or mutation due to substitution, insertion, deletion, or the like, and thus, it is possible to diagnose and prevent diseases accompanied by abnormal phosphate metabolism and calcium metabolism, or diseases developed into abnormal skeletal metabolism, or diseases developed into abnormal vitamin D metabolism.

It is currently reported, based on the results of linkage analysis of families with ADHR, that the gene responsible for ADHR is located at 12p13(Econs, M.J., et al, J.Clin.Invest.100: 2653-2657, 1997). In this report, the gene was also shown to be located in the 18cm range between the microsatellite markers D12S100 and D12S 397. We determined the location of the DNA of the invention by comparison with the reported location. The region of the DNA encoding the polypeptide of the present invention is the same as the region in which the gene responsible for ADHR is located. It is contemplated that the polypeptide of the present invention is encoded by a gene that causes ADHR, based on the biological activity of the polypeptide of the present invention and the location of the gene on the chromosome. This can be done by isolating cellular components from the blood of ADHR patients, isolating chromosomal DNA from the cellular components, and then looking for SEQ ID NO: 11 was confirmed by the mutation of the nucleotide sequence in the region indicated. Therefore, the gene containing the above nucleotide sequence region can be used as autosomal dominant hypophosphatemic rickets, X-linked hypophosphatemic rickets, hypophosphatemic bone diseases, osteoporosis, etc.

Between exons 1 and 2 of the DNA region encoding the polypeptide of the invention, there is an STS sequence which we have registered with NCBI Genbank under accession number G19259. This marker is considered to be very important in studying the relationship between DNA and genetic characteristics.

The present invention will greatly alter the traditional understanding of calcium metabolism, phosphate metabolism, bone metabolism and vitamin D metabolism. According to the present invention, it is possible to delay the arrival of the hemodialysis stage of chronic renal failure, or to provide a new treatment and diagnosis method for phosphate metabolism-related and calcium metabolism-related diseases, as well as metabolic bone diseases. In addition, the present invention may be used to improve or support existing treatments.

Brief Description of Drawings

FIG. 1 includes photographs showing amplification products analyzed by agarose electrophoresis. To study the tumor specificity of OST311, first strand cDNA extracted from tumor tissue and first strand cDNA extracted from control bone tissue were used as templates, and the sequences of SEQ ID NOs: 22 and 23 and the primers shown in SEQ ID NO: PCR was carried out using G3 PDH-specific primers shown in FIGS. 26 and 27.

FIG. 2 is a photograph showing that recombinant OST311 was detected by Western blotting of eluted fractions prepared by affinity purification of recombinant OST311 using a nickel resin followed by separation and purification using a strong cation exchange resin SP-5 PW.

Fig. 3A shows a polypeptide having SEQ ID NO: 2, which is suitable for use in making peptide antibodies using the computational functions of macvector version 6.5.1.

Fig. 3B shows the predicted expression of a polypeptide having seq id NO: 2, and (3) the hydrophobicity of the polypeptide of the amino acid sequence shown in the figure.

Fig. 4 shows the change in mean body weight over time between the tumor-free group (line represented by avr. -) and the group with tumors (line represented by avr. +) 31 days after transplantation of CHO-OST311H cells.

FIG. 5 includes several radiographs showing a full set of skeletal soft radiographs of tumor bearing individuals transplanted with control CHO ras clone 1 cells, tumor bearing individuals transplanted with CHO-OST190H cells, and tumor bearing individuals transplanted with CHO-OST311H cells.

Figure 6 shows the amino acid sequence alignment of human OST311 polypeptide and mouse OST311 polypeptide.

Fig. 7 shows the serum phosphate levels, serum calcium levels and serum alkaline phosphatase activity measurements for the tumor-free group (n ═ 6), the CHO ras clone-1 tumor group (n ═ 10), the CHO-OST190H tumor group (n ═ 10) and the CHO-OST311H tumor group-1 (n ═ 6) and the CHO-OST311H tumor group-2 (n ═ 6). Blood was collected from the heart of each individual on days 44 to 46.

FIG. 8 includes photographs showing a comparison of sodium-phosphate cotransporter (NaPi-7) expression levels as measured by Western blotting. Specifically, the brush-edged lamina membranes of proximal tubule epidermal cells were prepared from excised kidneys of CHO-OST311H tumor individuals and tumor-free individuals, and then the expression levels of NaPi-7 were compared by Western blotting.

FIG. 9A includes photographs showing changes in the mRNA levels of renal sodium-phosphate cotransporter (NaPi-IIb) as detected by Northern blotting. Kidneys were taken from mice sacrificed from day 44 to 46 after tumor transplantation.

FIG. 9B includes photographs showing changes in mRNA levels of sodium-phosphate cotransporter (NaPi-IIb) in mouse small intestine detected by Northern blotting. Small intestine was collected from mice sacrificed 44 to 46 days after tumor implantation.

FIG. 9C includes photographs showing the change in mRNA levels of mouse kidney vitamin D metabolizing enzyme (1. alpha. OHAse, 24OHAse) detected by Northern blotting. Kidneys were taken from mice sacrificed from day 44 to 46 after tumor transplantation.

Figure 10 shows X-ray pictures showing leg segments taken from mice sacrificed from days 44 to 46 after tumor implantation.

FIG. 11A includes a graph showing a comparison of serum phosphate levels and serum calcium levels at day 2 post-transplantation in nude mice (6 weeks old, BALB/c, male) transplanted with PBS, CHO ra s clone-1 cells and CHO-OST311H cells.

Each value is expressed as mean ± standard deviation.

FIG. 11B includes a graph showing a comparison of serum phosphate levels and serum calcium levels at day 6 post-transplantation in nude mice (6 weeks of age, BALB/c, male) transplanted with PBS, CHO ras clone-1 cells and CHO-OST311H cells.

Each value is expressed as mean ± standard deviation.

FIG. 12 includes photographs showing the results obtained by purifying CHO-OST311H cell culture supernatant and performing Western blotting of the eluted fractions using anti-His 6 antibody and anti-OST 311 peptide antibody (311-114). Left column display detects 311: 26-179, with 311: 25-179, right side 311: 180-251. Is marked with^*The fractions (in the upper half of the gel phase) were used for single dose testing on normal mice.

FIG. 13 includes photographs showing Villanueva-Goldner stained non-demineralized sections. The non-demineralized fraction was the proximal metaphyseal end of the tibia from mice transplanted with CHO-OST311H cells and tumor-free mice.

FIG. 14 includes photographs showing the results of Northern blotting of vitamin D metabolizing enzyme gene products in excised kidneys of mice transplanted with CHO-OST311H cells and control mice.

FIG. 15A shows the schedule for experiment 1 in which CHO-producing recombinant OST311H full-length protein was administered to normal mice. Fig. 15B includes graphs showing serum phosphate levels at each blood draw, and fig. 15C includes graphs showing serum calcium levels at the same time.

FIG. 16A shows the schedule for experiment 2 in which CHO-producing recombinant OST311H full-length protein was administered to normal mice. Fig. 16B includes graphs showing serum phosphate levels at each blood draw and fig. 16C includes graphs showing serum calcium levels at the same time.

FIG. 17 is a photograph showing recombinant proteins detected in the culture supernatant when Western blotting was performed on the culture supernatant of CHO-OST311RQH cells producing mutant recombinant OST311RQH and OST311RQH/pEAK rapid cells.

Figure 18A shows the time table of experiments in which mutant recombinant OST311RQH was administered to normal mice. Fig. 18B includes graphs showing serum phosphate levels at each blood draw, and fig. 18C includes graphs showing serum calcium levels at the same time.

FIG. 19A shows serum phosphate levels at day 2 post-transplantation in CHO-OST311RQH cell transplantation experiments. Figure 19B shows serum calcium levels in the same experiment.

FIG. 20 includes photographs showing recombinant OST311H detected by Western blotting in serum-free culture supernatant of CHO-OST311H cells using rabbit antisera raised against a partial peptide of OST 311.

Figure 21A is a table showing the levels of recombinant OST311 detected in each binding case when a sandwich ELISA was performed using 6 polyclonal antibodies against OST311 peptide.

FIG. 21B is a graph depicting the relationship between the concentration of purified recombinant OST311H and its corresponding measured value obtained using an ELISA system in combination with either 311-48 antibody or 311-148 antibody as the immobilized antibody and 311-148 antibody as the detection antibody.

FIG. 22A shows the expression levels of renal NaPi-7 as analyzed by Western blotting 1, 3, and 8 hours after administration of recombinant OST311 protein or vector to mice. FIG. 22B shows the expression level of renal NaPi-7 by Northern blot analysis using total renal RNA, following a similar procedure.

FIG. 23 shows the change in serum 1, 25-dihydroxyvitamin D3 levels at 1, 3 and 8 hours after administration of recombinant OST311 protein or vector to mice.

FIG. 24 shows expression of 25-hydroxyvitamin D-1-alpha-hydroxylase (1. alpha. OHAse) or 25-hydroxyvitamin D-24-hydroxylase (24OHAse) genes by Northern blot analysis using total renal RNA at 1, 3, and 8 hours after administration of recombinant OST311 protein or vector to mice.

FIG. 25 shows the average serum phosphate levels of each group when the average serum phosphate levels after 3 days of cell transplantation in the CHO-ras clone-1 cell transplant group were regarded as 100%.

FIG. 26 shows the nucleotide and amino acid sequences of recombinant His-OST311 encoded by plasmid OST311/pET28, and the DNA and amino acid sequences of recombinant MK-OST311 encoded by plasmid pET22-MK-OST 311.

FIG. 27 shows the elution pattern when recombinant refolded His-OST311 was purified by HPLC using cation exchange column SP-5PW (TOSOH, Japan).

FIG. 28 shows the elution pattern when performing HPLC purification of refolded MK-OST311 using cation exchange column SP-5PW (TOSOH, Japan).

FIG. 29 shows the elution pattern of PEG-modified recombinant MK-OST311 when purified by HPLC using cation exchange column SP-5PW (TOSOH, Japan).

FIG. 30 includes graphs showing serum phosphate levels at 8 or 9 hours after administration of (A) recombinant His-OST311 produced by E.coli or (B) recombinant MK-OST311 pegylated once.

FIG. 31 shows the results of Northern blot analysis of changes in expression of vitamin D metabolizing enzyme gene in kidney 1 and 4 hours after administration of His-OST311 recombinant produced by E.coli once.

FIG. 32 shows the change in serum levels of 1, 25-dihydroxyvitamin D3 1, 4 and 9 hours after one administration of E.coli-derived His-OST311 recombinants.

FIG. 33 shows mutant OST311 recombinants detected by Western blotting when mutations were introduced at amino acids 174 and 180 of OST311 and then the gene was expressed in pEAK cells to allow secretion of the mutant OST311 recombinants into the cell supernatant.

Best Mode for Carrying Out The Invention

The present invention is described in more detail by the following examples. These examples are not intended to limit the scope of the invention.

Example 1 construction of human tumor-induced osteomalacia-derived tumor cDNA library

Liquid nitrogen frozen tumor tissue was homogenized in 5ml ISOGEN (NIPPON GENE, Japan) solvent and then prepared to approximately 0.13mg total RNA according to the attached manufacturer's instructions. cDNA was synthesized from 1.5ul total RNA using SMART cDNA library preparation kit (CLONTECH, USA) according to the attached manufacturer's instructions. Hereinafter, the cDNA is referred to as cDNA # 2. Was this cDNA #2 ligated to an EcoRI linker and then inserted into the vector previously digested with the restriction enzyme EcoRI? ZAPII phage vector (STRATAGENE, USA). A tumor-induced osteomalacia tumor phage library was constructed using the Gigapack III Gold phage packaging kit (STRATAGENE, USA) according to the manufacturer's instructions attached. The resulting library contained a total of approximately 600,000 independent clones. Further, E.coli strain XLI-Blue MRF' was infected with the above phage library and then poured onto 20 plates (15 cm). These plates were incubated at 37 ℃ for 10 hours to form plaques. The whole plaque was aspirated into SM buffer to construct a tumor-induced osteomalacia tumor cDNA phage library.

Example 2 Positive screening protocol for tumor cDNA

Summary of the invention

The fact that tumor-induced osteomalacia can be cured by surgical resection of the tumor suggests that there may be a high and specific expression of the causative gene in the tumor. Furthermore, it has been reported that tumor-induced osteomalacia tumors so far are often composed of mesodermal, in particular mesenchymal cells. Therefore, it is necessary to identify a population of genes that are low in expression in normal mesoderm-derived tissues and are specifically and highly expressed only in tumor tissues. Therefore, positive screening was performed using the cDNA subtraction technique as described below. Tumor tissue-derived cDNA was subtracted from the cDNA isolated from the bone tissue as a control, so that genes that were not expressed in the bone tissue, but were specifically and highly expressed only in the tumor tissue, were enriched. The subtracted cDNA group is used as a probe to be hybridized with a tumor cDNA phage library, so as to obtain a gene segment specifically expressed in the tumor.

(1) Construction of cDNA for control human skeletal tissue

Liquid nitrogen frozen human skeletal tissue was homogenized in 5ml ISOGEN (NIPPON GENE, Japan) solvent and then prepared to about 0.011mg total RNA according to the attached manufacturer's instructions. cDNA was synthesized from 3ul total RNA using SMART cDNA library preparation kit (CLONTECH, USA) according to the attached manufacturer's instructions. Hereinafter, the cDNA is referred to as cDNA # 4.

(2) Subtraction of tumor-induced osteomalacia tumor cDNA and control bone tissue cDNA

To enrich for the highly expressed gene in the cDNA described in example 1, cDNA #2 and cDNA #4 described in example 2(1) were hybridized using the PCR-SelectcDNA subtraction kit (CLONTECH, USA) according to the attached manufacturer's instructions to subtract the gene fragment contained in cDNA #4 from cDNA # 2. The subtracted cDNA #2 was then amplified by PCR according to the instructions of the manufacturer, thus obtaining a subtracted cDNA set (A).

On the other hand, since the subtraction kit is characterized in that the hybridization process is performed only twice, which is less than that of the general technique, it is difficult to completely subtract a large amount of genes present in both materials by the kit. Accordingly, when a tumor cDNA library of tumor-induced osteomalacia is hybridized with the subtracted cDNA set (A) as a probe, genes that cannot be completely subtracted are also considered as positive clones. Therefore, the subtracted cDNA group (B) was prepared as a control probe by subtracting cDNA #2 from cDNA #4 described in example 2(1) in the same manner and then amplifying the subtracted cDNA #4 by PCR. The genes specifically contained in tumor-induced osteomalacia can be isolated by hybridizing the tumor cDNA library with the subtracted cDNA set (B) and the subtracted cDNA set (A) described above, respectively, and comparing the two signals.

(3) Differential hybridization of tumor cDNA library for tumor-induced osteomalacia

After infection of the E.coli strain XLI-Blue with the tumor cDNA phage library for tumor-induced osteomalacia described in example 1, the infected E.coli strain was re-inoculated to form 3000 plaques per plate (15cm), followed by culture at 37 ℃ for 8 hours. The plaques on each plate were then transferred to two Hybond N + (Amersham Pharmacia Biotech, USA) nylon membranes. The plaque-transferred nylon membrane was subjected to DNA immobilization treatment according to the instructions of the attached manufacturer, and then screened using the subtracted cDNAs (A) and (B) described in example 2(2), respectively, as probes.

Probe labeling, hybridization and signal detection were performed using the Alphos Direct system (Amersham Pharmacia Biotech, USA) according to the manufacturer's instructions. 100ng each of the subtracted cDNA (A) and cDNA (B) described in example 2(2) was used as a probe, which was then labeled with fluorescein according to the experimental protocol. The probes were added to hybridization solutions provided by 50ml Alphos Direct system, and at the same time, two groups of 8 plaque-transferred nylon membranes were hybridized and washed according to the protocol. After washing, the nylon membrane was subjected to a luminescence reaction, and ECL FILM (Amersham Pharmacia Biotech, USA) was exposed for 2 hours, developed with an automatic processor (FUJI FILM, Japan), and then analyzed for the result.

As a result, the independent plaques in the portion which were seriously scorched after exposure when cDNA (A) was used as a probe and not scorched when cDNA (B) was used as a probe were visually selected, scraped off from the plate, and then suspended in 0.5ml of SM buffer. The suspension was left at 4 ℃ for 2 hours or more to extract phages.

(4) Nucleotide sequence analysis of Positive clones

35 rounds of PCR were carried out using 0.5ul of the phage solution containing positive clones obtained in example 2(3) as a template, T7 primer (TAATACGACTCACTATAGGG) (SEQ ID NO: 24) and T3 primer (ATTAACCCTCACTAAAGGGA) (SEQ ID NO: 25) of the internal sequence of the phage vector, and LA-taq polymerase (TAKARA SHUZO, Japan), each cycle consisting of 96 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 30 seconds. The PCR product was subjected to 0.8% agarose gel electrophoresis. Clones that seen only a clear band were sequenced using ABI377 DNA sequencer (PE Applied systems, USA) using PCR amplified fragments as templates.

When two bands were seen, PCR products were extracted from the gels of the respective bands using QIAquick Gel Extraction kit (QIAGEN, Germany), and then sequenced by an ABI377 DNA sequencer.

As a result of differential hybridization of the plaques of 341000 tumor-induced osteomalacia phage libraries, 456 positive plaques were identified, and the nucleotide sequences of all these plaques were determined.

Example 3 narrowing of the range of human hypophosphatemia inducing factor candidate genes

A homology search was performed on the nucleotide sequences registered in Genbank (nucleotide sequence database provided by NCBI) using the sequence information of the 456 positive clones obtained in example 2. As a result, a group of genes shown in Table 1, which are existing genes having a high frequency of occurrence, was obtained. In addition, as a result of database search, 100 clones were unknown gene fragments whose biological activities were unknown. For nucleotide sequence information of these unknown gene fragments, overlap between clones is further abstracted as frequency information. Among them, the most frequently overlapping genes were obtained, including 7 cloned OST311 sequences that sequentially formed a contiguous sequence population. The nucleotide sequence obtained here was derived from SEQ ID NO: 1 from nucleotide 1522 to 2770. When a gene fragment of OST311 is searched in an existing database, it is not registered as cDNA or EST, but corresponds to a genomic sequence. The relevant genomic sequence was AC008012, which has been reported to be chromosomal at 12p 13. However, no protein-encoding region (ORF) was found in the obtained sequence, and therefore the sequence was expected to correspond to the 3' -untranslated region. Thus, the full-length cDNA was cloned from the tumor-induced osteomalacia cDNA library. Furthermore, the method is simple. The ORF was predicted using the DNASIS-Mac 3.7 version of ORF prediction function.

TABLE 1

Clone ID	Frequency of	Description of the invention
Clone ID	Frequency of	Description of the invention	OST131OST1OST2OST311OST1001OST584OST666OST133OST837OST562OST1002OST1003OST1004OST903	236351374332222222	Medullary protein 1(DMP1) Heat shock protein 90(HSP90) osteopontin unknown/genomic DNA12p13CD44 antigen fibronectin translation Regulation tumor protein Beta2 tubulin Fibroblast Growth Factor (FGF) annexin II/lipocortin II cytochrome oxidase subunit 2 cytokinin unknown

Example 4 cloning of full-Length OST311

According to the OST311 sequence obtained in example 3, the following primers were synthesized. Then, 35 PCR cycles were performed using the tumor-induced osteomalacia tumor cDNA library as a template, each cycle consisting of 96 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 30 seconds.

311-U65：TTCTGTCTCGCTGTCTCCC(SEQ ID NO：12)

311-L344：CCCCTTCCCAGTCACATTT(SEQ ID NO：13)

The PCR product was subjected to 2% agarose gel electrophoresis to confirm that the PCR product of the desired size was amplified, and then the PCR product was purified using a MicroSpin column S-300HR (Amersham pharmacia Biotech, USA). The resulting PCR products were fluorescently labeled using the Alphos Direct system (Amersham pharmacia Biotech, USA) according to the attached manufacturer's instructions. Then, 20000 clones of tumor-induced osteomalacia tumor cDNA library were plaque-hybridized using these labeled products as probes.

The 40 positive clones obtained were PCR amplified using T7 and T3 in the manner described in example 2 (4). Based on the nucleotide sequence of the resulting PCR product, primers 311-L296(SEQ ID NO: 14, GGGGCATCTAACATAAATGC) were synthesized. PCR products were obtained by amplification using 311-U65(SEQ ID NO: 12) and 311-L344(SEQ ID NO: 13) probes, and plaque hybridization was performed again on 20000 clones of the tumor-induced osteomalacia tumor cDNA library using these as probes. For the 62 positive clones, the nucleotide sequences of PCR products amplified using the T7 and 311-L296(SEQ ID NO: 14) primers were determined. The determined sequence is ligated to the already determined nucleotide sequence. Thus, SEQ ID NO: 1. It is clear that the ORF of OST311 starts at the position located in SEQ ID NO: 1 nucleotide 133. In addition, the following primers were synthesized in order to finally determine the sequence of ORF.

311-F1: AGCCACTCAGAGCAGGGCAC (SEQ ID NO: 15, nucleotides 112 to 131)

311-F2: GGTGGCGGCCGTCTAGAACTA (SEQ ID NO: 16, vector sequence)

311-F3: TCAGTCTGGGCCGGGCGAAGA (SEQ ID NO: 17, nucleotides 539 to 559)

311-L1: CACGTTCAAGGGGTCCCGCT (SEQ ID NO: 18, nucleotides 689 to 708)

311-L3: TCTGAAATCCATGCAGAGGT (SEQ ID NO: 19, nucleotides 410 to 429)

311-L5: GGGAGGCATTGGGATAGGCTC (SEQ ID NO: 20, nucleotides 200 to 220)

311-L6: CTAGATGAACTTGGCGAAGGG (SEQ ID NO: 21, nucleotides 868 to 888)

35 rounds of PCR were performed using 311-F2(SEQ ID NO: 16) and 311-L6(SEQ ID NO: 21) primers, a tumor-induced osteomalacia tumor cDNA library as a template, and Pyrobest DNA polymerase (TAKARA SHUZO, Japan), each cycle consisting of 96 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 30 seconds.

When the PCR product was subjected to 2% agarose gel electrophoresis, it was confirmed to be a fragment of about 980 nucleotide base pairs. The nucleotide sequence of the amplified fragment was then determined using the above primers (SEQ ID NOS: 15 to 21). The thus determined sequence encoding SEQ ID NO: 2(SEQ ID NO: 1) is located in the ORF region of the polypeptide shown in SEQ ID NO: 1 between the start codon ATG at nucleotide 133 and the stop codon TAG at nucleotide 886.

Example 5 specificity of OST311 against tumor-induced osteomalacia tumors

To investigate the tumor specificity of OST311, 35 rounds of PCR were performed using first strand cDNA extracted from tumor tissue and control bone tissue as a template, and OST 311-specific primers shown below (SEQ ID NOS: 22 and 23). Each round of PCR included 96 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 30 seconds. In addition, DMSO was added to each reaction solution to a final concentration of 2%, and LA-taq DNA polymerase (TAKARA SHUZO, Japan) was used as an enzyme. Next, as an internal standard, PCR was carried out under similar conditions using primers specific for G3PDH (FW: ACCACAGTCCATGCCATCAC (SEQ ID NO: 26), RV: TCCACCACCCTGTTGCTGTA (SEQ ID NO: 27)).

311F1EcoRI：CCGGAATTCAGCCACTCAGAGCAGGGCACG(SEQ ID NO：22)

311LHisNot：

ATAAGAATGCGGCCGCTCAATGGTGATGGTGATGATGGATGAACTTGGCGAA(SEQ ID NO：23)

These PCR products were subjected to 2% agarose gel electrophoresis as shown in FIG. 1. With the OST311 primer, PCR products of the expected size were observed only with tumor tissue as template. In contrast, when using the G3PDH primers, the same level of PCR product of the expected size was observed in both tumor tissue and control bone tissue. From these results, it was confirmed that OST311 is specifically expressed in tumor tissues.

Example 6 isolation of CHO cells stably expressing OST311

(1) Construction of OST311 expression vector

35 rounds of PCR were performed using the 311F1EcoRI (SEQ ID NO: 22) and 311 LHissNot (SEQ ID NO: 23) primers shown in example 5, with the tumor-induced osteomalacia tumor cDNA library as a template, each cycle (course) consisting of 96 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 30 seconds. In addition, DMSO was added to each reaction solution to a final concentration of 2%, and LA-taq DNA polymerase (TAKARA SHUZO, Japan) was used as an enzyme. The 311F1EcoRI primer anneals to the sequence of SEQ ID NO: 1, to nucleotide 111, 311LHisNot primer to SEQ ID NO: nucleotide 871 of 1. Encoding the amino acid sequence of SEQ ID NO: 2 can be amplified by PCR using these two primers. In addition, the 311LHisNot primer comprises a nucleotide sequence that is set forth in SEQ ID NO: 2 followed by 6 histidine residues and a stop codon after the last histidine codon. Thus, the translated recombinant protein has a His6 tag at the C-terminus, which can be used for antibody recognition of the recombinant, as well as purification of the recombinant using nickel resin.

After digestion with the restriction enzymes EcoRI and NotI, the PCR product was ligated into the plasmid vector pcDNA3.1Zeo (INVITROGEN, USA) similarly digested with EcoRI and NotI for expression in animal cells. The recombinant vector thus obtained was introduced into E.coli strain DH5 α. Escherichia coli was cultured in 3ml of LB medium containing 100mg/ml of ampicillin, and then the plasmid was purified using a GFX plasmid purification kit (Amersham pharmacia Biotech, USA). The sequence of the inserted gene is determined by conventional methods. Thus, it was confirmed that the sequence was identical to SEQ ID NO: 1 is identical, immediately preceding the stop codon a nucleotide sequence encoding the His6 tag sequence is added.

(2) Isolation of CHO cells stably expressing OST311

Approximately 20ug of a plasmid into which the OST311ORF portion prepared in example 6(1) was inserted was digested with the restriction enzyme FspI to cleave the site of the ampicillin resistance gene within the vector. The cut support was then ethanol precipitated and then dissolved in 10ul of ultrapure water. Subsequently, the entire volume of the solution was introduced into the host cells by electroporation using GenePulseII (BioRad, USA). CHO Ras clone-1 cells (Shirahata, S., biosci.Biotech.biochem, 59 (2): 345-347, 1995) were used as host cells. Cloning of CHO Ras-1 cells in 5% CO₂And 100% humidity, in a 75cm medium supplemented with 10% FCS MEM alpha²The flask was maintained until the cells grew to cover approximately 90% of the culture area. The attached cells were then detached by trypsin treatment, yielding approximately 1 × 10⁷A cell. The resulting cells were resuspended in 0.8ml PBS, mixed with FspI digested plasmid, and then cooled on ice for 10 minutes. The cells containing the plasmid were transferred into a 4 mm wide glass tube. After applying the electric pulses at the set values (0.25kV and 975uF), the glass tube was cooled for a further 10 minutes. The gene-transferred cells were cultured in MEM α medium containing 10% FCS for 24 hours. Then, Zeocin (INVITROGEN, USA) was added to the medium to a final concentration of 0.5mg/ml, followed by further culture for 1 week. Subsequently, for the cloned cells showing drug resistance, these cells were reseeded in a 96-well plate by the limiting dilution method at 0.2 cells/well, and then cultured for about 3 weeks in the presence of zeocin at a final concentration of 0.3mg/ml, thereby obtaining 35 clones of drug-resistant strains.

(3) Demonstration of recombinant production by CHO cells stably expressing OST311

The presence of recombinant OST311 in the conditioned medium was confirmed by Western blotting for 35 clones showing drug resistance.

The collected 0.2ml conditioned medium was concentrated to about 40 to 50ul using an ultra-free MC M.W.5000 cut-off membrane system (MILLIPORE, USA). To the concentrate was added 10ul of a sample buffer containing 1M Tris-C1pH6.8, 5% SDS, 50% glycerol and 100mM DTT, followed by heating at 95 ℃ for 5 minutes. The proteins in the conditioned medium were then separated by polyacrylamide electrophoresis with a gradient of 10 to 20%. Thereafter, the proteins in the gel were transferred to Immobilon PVDF membrane (MILLIPORE, USA) using a semi-dry blotting system (Owlseparation Systems, USA). The PVDF membrane and in TTBS buffer (Sigma, USA) 1/5000 diluted anti His (C-terminal) antibody (INVITROGEN, USA) together with room temperature 1 hours incubation. Then, the membrane was exposed to light for 5 minutes using an ECL system (Amersham pharmacia biotech, USA) and developed with an automatic processor (FUJIFILM, Japan). As a result, clone #20 was isolated, with the strongest signals at approximately 32kDa and 10 kDa. Thereafter, #20 cells were named CHO-OST311H and deposited at national institute of advanced Industrial science and technology, International patent organism depositary (Higashi, Tsukuba-shi, Ibaraki 1-1-1) (accession number FERM BP-7273).

Example 7 measurement of phosphate uptake inhibitory Activity of CHO-OST311H

Conditioned medium

CHO-OST311H at 37 ℃ in 5% CO₂And 100% humidity, in a 225cm medium containing 10% FCS supplemented MEM alpha²The flask was incubated until the cells grew covering approximately 80% of the flask area. Then, the medium was replaced with 30ml of serum-free medium CHO-S-SFM II (LIFETECHNOLOGY, USA). After 48 hours, the conditioned medium was collected. The conditioned medium was centrifuged at 1200g for 5 minutes to remove suspended cells and the like, and then filtered with a Minisart-plus 0.22um filter (Sartorius, Germany).

Using this conditioned medium, its effect on the phosphate uptake activity of the human renal proximal tubule cell line (CL-8 cells) was examined. Said personRenal proximal tubule cell line at 37 ℃ in 5% CO₂And 100% humidity in DMEM medium containing 10% FCS (LIFE TECHNOLOGY). To measure the phosphate uptake activity, the human kidney proximal tubule cell line was first cultured in 48-well plates of DMEM medium (CORNING, USA) containing 10% FCS. When the cells grew to cover the entire bottom surface of the plate after 3 days from the start of the culture, the medium was replaced with 200ul of serum-free medium CHO-S-SFM II (LIFE TECHNOLOGY, USA), followed by further culturing for 20 to 24 hours. The following experiment was performed with cells in this state to measure phosphate uptake activity (experiments 1 and 2).

(1) Experiment 1:

the CHO-S-SFM II medium was removed and 200ul of the conditioned medium of CHO-OST311H cells, as described above, prepared in CHO-S-SFM II was added to each well. As control wells, 3 wells containing no medium in place of CHO-S-SFM II, and 3 wells containing medium supplemented with 200ul of conditioned medium from CHO-OST190H cells prepared in a similar manner as CHO-OST311H, respectively. The CHO-OST190H cell was obtained by introducing OST190H into CHOras clone-1 to express OST190H in a similar manner to the preparation of CHO-OST311H cell. Similar to OST311H, CHO-OST190H was expressed containing a His6 tag sequence added to the C-terminus of the polypeptide, which is identical to the polypeptide reported by Rowe, P.S.N et al (Genomics 67: 54-68, 2000) under the name MEPE. After addition of each sample, in CO₂After culturing in the incubator for another 26 hours, the phosphate uptake activity of the cells in each well was measured by the following method for measuring the phosphate uptake activity.

(2) Test 2:

100. mu.l of the culture broth was aspirated from 200. mu.l of the CHO-S-SFMII medium being cultured. For these cultures, 100. mu.l of conditioned medium from CHO ras clone-1 cells were added to 3 wells, respectively, and 100. mu.l of conditioned medium from CHO-OST311H cells were added to 3 wells, respectively. Then, in CO₂The culture was carried out in an incubator for 24 hours. Subsequently, the phosphorus of the cells in each well was determined by the following method for measuring the phosphate transport activityAcid salt uptake activity.

Measurement of phosphate uptake activity:

after addition and incubation of conditioned medium, phosphate-free buffer (150mM NaCl, 1mM CaCl) was used₂1.8mM MgSO4, 10mM HEPES, pH7.4), and then incubated in the same solution at room temperature for 10 minutes. Aspirating the solution and adding an assay solution by adding radioactive KH to the buffer₂PO₄(NEN) prepared to a concentration of 0.105 mM. The solution was incubated at room temperature for 10 minutes. After incubation, the assay was aspirated off immediately with stop solution (150mM CaCl) that had been frozen₂，1.8mM MgSO₄10mM HEPES, pH7.4) were washed 3 times. The washing solution was aspirated, and 80. mu.l of 0.2N NaOH was added to the cells, followed by incubation at room temperature for 10 minutes, thereby lysing the cells. To determine the radioactivity in the cell lysates, the solutions were transferred to ReadyCap (Beckman), dried at 50 ℃ and then placed in glass tubes. Then, the radioactivity was measured by a scintillation counter (Wallac1410, Pharmacia). The phosphate uptake activity in each assay is shown in table 2, where the average uptake activity of the control without supplemented conditioned medium is taken as 100%. The conditioned medium of CHO-OST311H cells significantly inhibited the phosphate uptake activity of human kidney proximal tubular epidermal cells.

TABLE 2 Activity of OST311 against phosphate uptake by renal tubular epidermal cells

Test 1

Example 8 partial purification of recombinant OST311 from CHO-OST311H conditioned Medium

Recombinant OST was partially purified from conditioned medium prepared as described in example 7 by the following method. Processes 1) and 4) were carried out in a chromatography chamber at 4 ℃.

1) A degradable polypropylene column (INVITROGEN, USA) was packed with ProBond nickel resin to make the bed volume 3ml, then washed and equilibrated with 30ml buffer 1 (Table 3).

2) 120ml of conditioned medium prepared in the manner described in example 7 was loaded by free descent into the above nickel resin column to bind recombinant OST 311.

3) 30ml of buffer 2 shown in Table 3 was used to remove non-specifically adsorbed proteins.

4) 3ml of the buffer solution shown in Table 3 was added in 4 portions to elute recombinant OST 311. 20ul of each of these 4 fractions were directly subjected to Western blotting in the manner described in example 6 (3). Thus, detection of OST311 was attempted with an anti-His antibody.

As a result, intense signals of approximately 32kDa and 10kDa were observed in the second fraction.

5) The second fraction was loaded onto NAP25 and NAP10 columns (Amersham pharmacia Biotech, USA) and the solvent was replaced with buffer 4 shown in Table 3.

6) Recombinant OST311, which had replaced the solvent with buffer 4, was loaded onto SP-5PW (Strong cation exchange resin, TOSOH, Japan) at a flow rate of 1 ml/min using high performance liquid chromatography (Hitachi, Japan). Elution was performed by adding buffer 5 shown in Table 3 at a gradient of 1%/min, so that 2ml was collected from each fraction. As shown in fig. 2, Western blots were performed on each eluted fraction in the manner described in example 8(5) to attempt to detect OST 311. The approximately 10kDa signal elutes with about 280mM NaCl and the approximately 32kDa signal elutes with about 400mM NaCl. The corresponding fractions were subjected to SDS polyacrylamide gel electrophoresis, followed by staining with a silver staining kit (Daiichi Chemicals, Japan). Fractions containing about 10kDa and 32kDa signals were 70% pure or more.

TABLE 3

Buffer solution 1	Buffer solution 2	Buffer solution 3	Buffer 4	Buffer solution 5
Buffer solution 1	Buffer solution 2	Buffer solution 3	Buffer 4	Buffer solution 5	10mM Na/PipH6.50.5M NaCl	10Mm Na/PipH6.510mM imidazole 0.5M NaCl	10Mm Na/PipH6.510mM imidazole 0.5M NaCl5mMCHAPS	10Mm Na/PipH6.55mMCHAPS	10mMNa/PipH6.51M NaCl5mMCHAPS

Na/Pi: sodium phosphate buffer solution

Example 9N-terminal amino acid sequence analysis of partially purified recombinant OST311

Approximately 10kDa and 32kDa signals recognized by anti-His antibodies in the partially purified fractions obtained by the method described in example 8 were subjected to SDS polyacrylamide gel electrophoresis. Subsequently, the proteins in the gel were transferred to Immobilon PVDF membrane (MILLIPORE, USA) using a semi-dry blotting System (Owl Separation System, USA). The PVDF membrane was stained with CBB, bands of approximately 10kDa and 32kDa were cut off, and the N-terminal amino acid sequence was determined by means of a protein sequencer model 492 (PE Applied Systems, USA).

As a result, it was apparent that the N-terminal amino acid sequence of the band of about 35kDa was represented by SEQ ID NO: 2, the OST311 sequence starting at residue 25 Tyr. From this result, it was confirmed that the sequence of the amino acid sequence shown in SEQ ID NO: 2 from the first residue Met to the 24 th residue Ala as a secretion signal sequence. In another aspect, the N-terminal amino acid sequence of the about 10kDa band is a sequence selected from SEQ ID NO: 2, residue 180Ser, and the OST311 sequence. The presence of a motif containing RRXXR immediately before residue 180Ser indicates that recombinant OST311 is cleaved by some CHO cell-produced proteases.

As described above, recombinant OST311 produced by CHO-OST311 cells is present as at least 3 polypeptides after secretion: a polypeptide from residues 25Tyr to 251Ile (SEQ ID NO: 4), a polypeptide from residues 25Tyr to 179 Arg (SEQ ID NO: 6), and a polypeptide from the sequence SEQ ID NO: 2 from residue 180Ser to 251Ile (SEQ ID NO: 8).

EXAMPLE 10 preparation of polyclonal antibody against OST311 partial peptide

The calculation function of macvector6.5.1 version was used to predict SEQ ID NO: 2, thereby inferring the antigenic sites suitable for the production of peptide antibodies (fig. 3A and B). Here, suitable sites are predicted from the viewpoint that sites having strong hydrophobicity are not easily subjected to sugar chain modification and phosphorylation. Thus, 311-48(SEQ ID NO: 28) was selected and synthesized as the antigen by, during the synthesis phase, giving a peptide derived from the sequence SEQ ID NO: 2 by artificially adding a cysteine residue to the C-terminus of the 20 amino acid-containing peptide starting at residue 48Arg, and 311-114(SEQ ID NO: 29) similarly prepared by adding a cysteine residue to the 20 amino acid-containing peptide starting at residue 114 Arg. Specifically, cysteine residues were artificially added to the C-termini of the two peptides during the synthesis phase so that the product could be coupled to a carrier protein (bovine thyroglobulin). The immunization of rabbits with coupled carrier proteins was delegated to IBL, CO., Ltd. (1091-1, Fujioka-shi, Gunma, Japan) (delegation No.: 1515).

311-48：RNSYHLQIHKNGHVDGAPHQC(SEQ ID NO：28)

311-114：RFQHQTLENGYDVYHSPQYHC(SEQ ID NO：29)

Example 11 experiment of transplantation of CHO-OST311H cells into nude mice

To examine whether OST311 is a causative agent of tumor-induced osteomalacia, CHO-OST311H cells were transplanted into 6-week-old BALB/c nude mice (males) to induce tumors, thereby establishing a murine tumor-induced osteomalacia model that continuously secretes recombinant OST311 from the tumors. As an experimental control, CHO ras clone-1 cells and CHO-OST190H described in example 7 were used similarly in the transplantation experiment.

(1) Transplantation of CHO cells

CHO-OST311H cells were detached from flasks at 1X10 by trypsinization⁸Cells/ml were suspended in PBS. Two latera (2X 10) were injected subcutaneously into nude mice with 0.1ml each of the suspension⁷Cell/mouse). In addition, as a control group, the same number of CHO ras clone-1 cells were injected subcutaneously in the same manner. Approximately 1 month after injection, 5 nude mice were housed in a plastic cage and allowed to contact the solid food CE-2 (CLEAAJAPAN, Japan) and tap water, ad libitum. Two weeks after transplantation, tumorigenesis was observed in 75% of the control group and 66.7% of the OST311 group.

(2) Comparison of body weight changes

After transplantation of CHO-OST311H cells, the mean body weight change over 31 days was compared between the tumor-free group (line indicated by avr. -) and the CHO-OST311H cell tumor group (line indicated by avr. + in the graph). As shown in FIG. 4, the CHO-OST311H cell tumor group showed an inhibition of body weight gain compared to the tumor-free group, and a significant difference between the two groups (24.1. + -. 1.5g vs. 26.7. + -. 1.0g, p < 0.001, day 31). In contrast, no similar difference was seen in the mean body weight between the CHO ras clone-1 cell tumor group and the tumor-free group (27.0 ± 1.8g vs.26.7 ± 1.0g, no significant difference, day 31).

(3) Measurement of serum phosphate and calcium, and urine phosphate and calcium

Nude mice were housed in metal cages for 24 hours 30 and 40 days after cell transplantation. After collecting urine, blood was collected from the heart or the eyebox of the nude mouse under the anesthesia of ether (diethyl ether). Sera were prepared from peripheral blood using a Microtainer (Beckton Dickinson, USA). After measuring the volume, the urine was centrifuged to collect the supernatant. Serum and urine phosphate levels were measured using P-test Wako (Wako Pure Chemical Industries, Japan), serum and urine calcium levels were measured using calcium-test Wako (Wako Pure Chemical Industries, Japan), and serum and urine creatinine levels were measured using CRE-EN KAINOS (KAINOS, Japan).

Test 1:

on day 34 after cell transplantation, serum phosphate levels were measured for the tumor-free group, the CHO ras clone-1 cell tumor group, the CHO-OST190H cell tumor group, and the CHO-OST311H cell tumor group.

Test 2: serum and urine phosphate levels, calcium levels and creatinine levels were measured for the tumor-free group and the CHO-OST311H cell tumor group from day 44 to 46 after cell transplantation. Phosphate or calcium clearance was divided by creatinine clearance to determine renal partial excretion of phosphate and calcium.

The measurement results are shown in table 4 below.

Table 4 serum and urine phosphate and calcium in cell-transplanted mice

Experiment 1

Experiment 2

(4) Soft X-ray photograph of complete skeleton

After transplantation of CHO-OST311H cells, it was confirmed that tumors had formed, and individuals showed significant abnormalities in physical condition and walking function as compared with tumor-free individuals or control CHO ras clone-1 transplanted individuals. Therefore, the individual with the tumor is expected to develop skeletal abnormalities. Then, individuals believed to have developed tumors were randomly selected from the control CHO ras clone-1 transplanted group, the CHO-OST190H cell transplanted group and the CHO-OST311H cell transplanted group, and radiographs were taken using the radiography system uFX-100(FUJI FILM, Japan) according to the attached manufacturer's instructions. The conditions for taking the X-ray photograph were an X-ray tube voltage of 25kV, a current of 0.1mA, and an exposure time of 10 seconds. The individuals were exposed to an imaging plate and then subjected to image analysis using BAS2000(FUJI FILM, Japan).

As a result, as shown in FIG. 5, the brightness of soft X-ray photographs of the whole paraskeleton of CHO-OST311H cell-transplanted individuals was decreased, thereby confirming that mineralization was deficient. In addition, skeletal deformities, such as deformation of the costal cage, were also verified.

(5) Determination of serum phosphate and calcium levels and alkaline phosphatase activity

Blood was collected from the heart on days 44 and 46 after cell transplantation, and serum obtained therefrom was stored at-20 ℃. Serum samples were thawed together and the phosphate and calcium levels in each serum were again measured while alkaline phosphatase activity was determined. Phosphate levels were measured using P-test Wako (Wako Pure Chemical Industries, Japan), calcium levels were measured using calcium-test Wako (Wako Pure Chemical Industries, Japan), and alkaline phosphatase activity was measured using calcium alkaline phosphorus B-test Wako (Wako Pure Chemical Industries, Japan). The results were divided into a tumor-free group (n-6), a CHO tumor group (n-10), a CHO-OST190H cell tumor group (n-10) and a CHO-OST311H tumor group (n-6 x 2). The CHO-OST311H components were divided into two groups: one group was sacrificed on day 44 (CHO-OST 311H-1: n-6) and one group was sacrificed on day 46 (CHO-OST 311-2: n-6). As shown in FIG. 7, significant changes were seen in the CHO-OST311H tumor group, including decreased serum phosphate levels (FIG. 7A), decreased serum calcium levels (FIG. 7B), and increased serum alkaline phosphatase activity (FIG. 7C).

(6) Expression of sodium-phosphate cotransporter protein (NaPi-7) in the proximal tubule of kidney

i) Preparation of proximal tubule epidermal cell brush border membrane (hereinafter BBM)

The kidneys were excised from CHO-OST311H tumor individuals and tumor-free individuals anesthetized with diethyl ether. Each kidney was cut in half to obtain coronal sections (experiment 1: 6CHO-OST 311H tumor individuals and 4 tumor-free individuals. experiment 2: 6CHO-OST 311H tumor individuals and 2 tumor-free individuals). BBM was prepared according to the protocol reported by Kessler et al (biochem. Biophys. acta.506, 136-154) using each half of the kidney excised from each individual.

The kidney was homogenized in 3ml of a homogenization buffer (50mM mannitol, 2mM Tris/HEPES pH7.5) with a glass homogenizer at 1300rpm for 2 minutes to obtain a homogenized kidney extract. Adding CaCl into the extract₂After reaching a final concentration of 10mM, the solution was stirred at 4 ℃ for 15 minutes and then centrifuged at 4900g at 4 ℃ for 15 minutes. The resulting supernatant was filtered with a Kimwipe and then centrifuged at 16200g for 60 minutes at 4 ℃ to precipitate a fraction containing a large amount of BBM. The pellet was resuspended in 5ml of suspension buffer (50mM mannitol, 2mM Tris/HEPES pH7.5), and the solution was centrifuged again at 16200g for 60 minutes at 4 ℃. This procedure was repeated twice, and the product was then resuspended in 0.1ml of suspension buffer. The protein concentration of the solution thus obtained was determined by standard methods to be 3 to 4 mg/ml.

ii) Western blotting of BBM proteins

The BBM protein from each mouse was diluted to 10ug/ul with suspension buffer as described above. Then, 2.5ul of a sample buffer containing 1M Tris-Cl, pH6.8, 5% SDS, 50% glycerol and 100mM DTT was added to the diluted solution. After heating the solution at 95 ℃ for 5 minutes, proteins in the BBM solution were separated by polyacrylamide electrophoresis with a gradient of 10 to 20%. Subsequently, the proteins in the gel were transferred to Immobilon PVDF membrane (MILLIPORE, USA) using a semi-dry blotting system (Owl separation systems, USA). The PVDF membrane was incubated with 1/2000 anti-NaPi-7 polyclonal antibody diluted in TTBS buffer (Sigma, USA) for 3 hours at room temperature. The antibody is a polyclonal antibody obtained by immunizing rabbits with a synthetic peptide corresponding to the C-terminal site of mouse NaPi-7 by a standard method of KIRIN EREWERYCO, LTD, Pharmaceutical Research Laboratories, Pharmaceutical division. After reacting with the antibody, the reaction product was further incubated with an anti-rabbit IgG secondary antibody (DAKO, Denmark) conjugated with horseradish peroxidase (HRP), and then the band was detected with an ECL system (Amersham pharmacia Biotech, USA).

Under reducing conditions, the antibody detected bands of approximately 80kDa and 35kDa and fragmented bands of macromolecules ranging from 170kDa to 200kDa (FIG. 8). These band patterns are the same as reported by Tatsumi et al (J.biol.chem 273, 28568-28575, 1998), and these bands are shown to vary uniformly with the amount of phosphate taken up from the food by the mouse or rabbit. From these facts, it was confirmed that the band was a polypeptide derived from NaPi-7. As shown in FIG. 8, for all of the above fragments (bands marked with arrows), the BBM protein from the CHO-OST311H tumor individuals contained a significant reduction in NaPi-7 signal as compared to that from tumor-free individuals. These results can be repeated in experiments 1 and 2, respectively. On the other hand, these BBM proteins were separated by polyacrylamide electrophoresis with a gradient of 10 to 20% and then stained with CBB. BBM protein from each individual was equally stained, indicating that the signal drop in Western blots is characteristic of NaPi-7 (FIG. 8). From this fact, it was concluded that OST311 protein acts on renal proximal tubule cells to down-regulate the expression of NaPi-7 at the protein level, thereby inducing hypophosphatemia.

(7) mRNA alteration analysis of phosphate Carrier protein and vitamin D metabolizing enzymes in the Kidney and Small intestine

i) Preparation of Total RNA

The small intestine and kidney were excised from mice sacrificed from day 44 to 46 after cell transplantation. The kidneys were quickly frozen in dry ice. The frozen kidney is stored in a low temperature freezer at-80 ℃ for use. A frozen kidney was homogenized in 5ml ISOGEN (Nippon Gene, Japan), and total RNA was prepared according to the attached manufacturer's instructions. 15ug of total RNA was electrophoresed through a 1% agarose formaldehyde-containing denaturing gel according to standard procedures and then transferred by capillary transfer overnight onto Hybond-N + (Amersham Pharmacia, USA). The membrane to which the RNA was transferred was irradiated with UV, and the transferred RNA was fixed using Stratakinker (STRATAGENE, USA), washed with 2XSSC, air-dried, and then stored at room temperature for use. The small intestine was washed with physiological saline to remove the contents, and then turned over. Subsequently, the small intestine epidermis was harvested by scraping with a formulation and then flash frozen with liquid nitrogen. And refrigerating the frozen small intestine epidermis in a refrigeration house at the ultralow temperature of minus 80 ℃ for standby. Frozen small intestine epidermis was homogenized in 5ml of ISOGEN (Nippon Gene, Japan), and total RNA was prepared according to the instructions of the attached manufacturer. 20ug of total RNA was electrophoresed through a 1% agarose formaldehyde-containing denaturing gel according to standard procedures and then transferred by capillary transfer overnight onto Hybond-N + (Amersham pharmacia, USA). The membrane to which the RNA was transferred was irradiated with UV, and the transferred RNA was fixed using Stratalinker (STRATAGENE, USA), washed with 2XSSC, air-dried, and then stored at room temperature for use.

ii) preparation of template DNA for Probe

5ug of total RNA prepared from a mouse (mouse 1) was added to 20ul of a reaction solution (50mM Tris (pH8.3), 75mM KCl, 3mM MgCl₂10mM DTT, 25g/ml (dT)18, 2.5mM dNTP, 200 units of MMLV reverse transcriptase (TOYOBO, Japan)) at 37 ℃ for 1 hour to synthesize cDNA, and then the reaction solution is incubated at 70 ℃ for 15 minutes to inactivate the enzyme. The synthesized cDNA was diluted 5-fold and used in the following reaction.

The following primers were synthesized from sequences registered in GenBank (NCBI, USA) and then used in PCR reactions:

synthetic primers for obtaining mouse GAPDH cDNA

mGAPDHFW TGAAGGTCGGTGTGAACGGATTTGGC(SEQ ID NO：30)

mGAPDHRV CATGTAGGCCATGAGGTCCACCAC(SEQ ID NO：31)

Synthetic primer for obtaining mouse Npt-1 cDNA

mNPt1FW GTAAAGAACCCTGTGTATTCC(SEQ ID NO：32)

mNpt1RV CTGCCTTAAGAAATCCATAAT(SEQ ID NO：33)

Synthetic primer for obtaining mouse NaPi-7cDNA

mNaPi7FW GAGGAATCACAGTCTCATTC(SEQ ID NO：34)

mNaPi7RV CTTGGGGAGGTGCCCGGGAC(SEQ ID NO：35)

Synthetic primer for obtaining mouse NaPi-2b cDNA

mNaPi2bFW TCCCTCTTAGAAGACAATACA(SEQ ID NO：36)

mNaPi2bRV GTGTTTAAAGGCAGTATTACA(SEQ ID NO：37)

Synthetic primer for obtaining mouse vitamin D1 alpha hydroxylase cDNA

m1 a OHaseFW CAGACAGAGACATCCGTGTAG(SEQ ID NO：38)

m1 a OHaseRV CCACATGGTCCAGGTTCAGTC(SEQ ID NO：39)

Synthetic primer for obtaining mouse vitamin D24 hydroxylase cDNA

m24OHaseFW GACGGTGAGACTCGGAACGT(SEQ ID NO：40)

m24OhaseRV TCCGGAAAATCTGGCCATAC(SEQ ID NO：41)

The reaction solution was prepared according to the instructions of the manufacturer attached to TakalA LA-Taq (TAKARA SHUZO, Japan). Mix 1 μ l cDNA and each 10pmol of the above primer were added to 50ml of the reaction solution. The solution was held at 94 ℃ for 1 minute and then subjected to 40 rounds of amplification, each incubation cycle comprising 94 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 1 minute. The amplified bands were then separated by 0.8% agarose gel electrophoresis, and the target fragment was recovered using Gene Clean II (Bio101, USA). For GAPDH, using the fragment obtained above as a template and a Megaprimer labelling kit (Amersham Pharmacia Biotech, USA), preparation³²P-labeled probe, then used for the following hybridization. For other genes, the resulting PCR fragment was ligated to pGEM-T vector (Promega, USA), and then introduced into E.coli DH 5. alpha. 10pmol each of T7(SEQ ID NO: 42) and SP6 primer (SEQ ID NO: 43) was added to the PCR reaction solution, and then transformed E.coli was also added. After the solution was held at 94 ℃ for 10 minutes, 40 rounds of amplification were performed, each cycle consisting of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute. The amplified bands were separated from the reaction solution by 0.8% agarose gel electrophoresis, and the target fragment was recovered by Gene CleanII (Bio101, USA).

T7 TAATACGACTCACTATAGGG(SEQ ID NO：42)

SP6 GATTTAGGTGACACTATAG(SEQ ID NO：43)

The nucleotide sequence of the amplified fragment obtained by the above procedure was determined using an ABI377 DNA sequencer (PE Applied System, USA), thereby confirming that each target fragment was obtained. The resulting DNA fragment was digested with Megaprimer Labeling kit (Amersham Pharmacia, USA)³²P-labeled and then used as a probe for the following hybridization process.

iii) hybridization

Hybridization was carried out using either ExpressHyb hybridization solution (CLONTECH, USA) or PerfectHyb hybridization solution (TOYOBO, Japan) according to the attached manufacturer's instructions. After hybridization and washing, the imaging plate was exposed for 30 minutes to overnight and then analyzed with BAS2000 image analyzer (FUJIFILM, Japan) (fig. 9A to C). In addition, the signal intensity of the target band was measured. After correcting the signal intensity of each gene with the signal intensity of GAPDH, the ratio of the mean of the tumor-free group (mice 1 to 4) to the tumor group (mice 5 to 10) was obtained. Table 5 below shows the ratios. With tumor formation in the group transplanted with CHO-OST311H, the mRNA level of NaPi-7, type II phosphate carrier protein in the kidney decreased significantly, while there was no major change in NPT-1, type I phosphate carrier protein in the kidney. In addition, there was a significant reduction in the mRNA for the NaPi-IIb and phosphate carrier proteins in the small intestine. In contrast, for the renal vitamin D metabolizing enzymes, the mRNA for both 25-hydroxyvitamin D-1-alpha hydroxylase (1. alpha. OHAse) and 25-hydroxyvitamin D-24-hydroxylase (24OHAse) was increased.

TABLE 5 mRNA ratios of tumor vs. tumor-free groups

(tumor group/non-tumor group)

NPT-1	0.88
NPT-1	0.88	NaPi-7	0.50
NaPi-2b	0.23	NaPi-7	0.50
NaPi-2b	0.23	Vitamin D1 alpha hydroxylase	3.90
Hydroxyvitamin D24 hydroxylase	1.94	Vitamin D1 alpha hydroxylase	3.90

(8) Measurement of serum 1, 25-dihydroxyvitamin D levels

Equivalent amounts of serum were collected from each control group mouse and the OST311H group mouse at day 44 and 46 after tumor implantation. Serum collected from each group (each group amounting to 0.5ml) was submitted to Mitsubishi Kagaku Bio-Clinical Laboratories, Inc, and then the level of 1, 25-dihydroxyvitamin D contained in the serum was measured in a manner similar to the Clinical test. As a result, the serum 1, 25-dihydroxyvitamin D levels of the control group and OST311 group were 28.0pg/ml and 23.9pg/ml, respectively. As described above, even in the case of hypophosphatemia and hypocalcemia, the level of 1, 25-dihydroxyvitamin D was not increased. This result clearly indicates that the effect of vitamin D metabolism is due to the action of OST 311.

(9) Soft X-ray photograph of leg joint

Tumor-free mice as well as CHO ras clone-1, CHO-OST190H or CHO-OST 311H-transplanted mice were sacrificed at day 44 to 46 after transplantation, leg segments were collected and then fixed in 4% neutral formalin for 3 days. Next, the soft tissue surrounding the bone is removed. Two were randomly selected from each group, and then X-rays were irradiated using a radiography system uFX-100(FUJIFILM, Japan) under the following conditions: the X-ray tube voltage was 25kV, current 0.1mA, exposure time 5 seconds, and then the imaging plate was exposed. The results are shown in FIG. 10. A reduction in the bone pillar of cortical bone (bone trabecula of the corticaldone) was observed in the CHO-OST311H group.

Example 12 analysis of OST311 nucleotide sequence homology and genomic region

Using SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 1, a molecule corresponding to OST311 can be retrieved from a plurality of species. Using the nucleotide sequence of SEQ ID NO: 1, the genome sequence database of mice was searched, and thus a sequence having a very high homology with OST311 was found in the sequence of mouse chromosome 6, which was deposited in Genbank under accession number AC 015538. The amino acid sequence of a partial polypeptide of mouse OST311 obtained by the sequence is shown as SEQ ID NO: 10, and the nucleotide sequence corresponding to the partial cDNA sequence is shown as SEQ ID NO: shown at 9. Fig. 6 shows the results of comparison of amino acid homology between human OST311 polypeptide and mouse OST311 polypeptide. As shown in example 11, it is apparent that the OST311 polypeptide having a human amino acid sequence has significant biological activity in mice. These results indicate that the activity can be easily retained even if those amino acids in the region of low homology in the amino acid sequence shown in FIG. 6 are substituted, deleted or inserted.

We compared SEQ ID NO: 1 and human 12p13 BACRPCI11-388F6 (accession number AC008012), which was found by using a database to have a region that is homologous to the nucleotide sequence shown in SEQ ID NO: 1, so that the sequence of the region encoding OST311 is determined. The nucleotide sequence adjacent to the OST311 gene is shown in SEQ ID NO: a TATAA box located in SEQ ID NO: 11 from nucleotide 498 to 502. The first sequence matching the cDNA sequence (SEQ ID NO: 1) is selected from the group consisting of SEQ ID NO: 11 begins at nucleotide 1713 and continues to nucleotide 2057. And SEQ ID NO: 1 is selected from seq id NO: 11 from nucleotide 8732 to 8833. This portion is considered exon 2. And SEQ ID NO: 1 begins with SEQ ID NO: 11, nucleotide 10644, and ends at nucleotide 12966. SEQ ID NO: 11 from nucleotide 498 to 12966 may be considered to be at least part of the gene encoding OST 311. Furthermore, it is evident that the STS sequence registered as G19259 in Genbank is located between exons 1 and 2. The OST311 is located in the region of 12p 13. From the results of the linkage analysis it can be concluded that the gene responsible for the autosomal vitamin D rickets (ADHR) is located in the 18cM region between D12S100 and D12S397, i.e. the microsatellite marker of 12p13 (in particular a region of about 10cM between D12S314 and D12S 397), according to Econs, M.J. et al (J.Clin.invest.100: 2653-2657, 1997). We evaluated the physical location of OST311 and the microsatellite marker of chromosome 12 above. As a result, D12S100 and D12S314 are within a 4602 to 6129kb region, OST311 is within an 8958 to 9129kb region, and D12S397 is within a 16280 to 16537kb region. Based on these results and the strong phosphate metabolism-regulating activity of OST311, we found that OST311 is a gene responsible for ADHR.

Example 13 short-term experiment of transplantation of CHO-OST311H cells into nude mice

CHO-OST311H cells were transplanted subcutaneously into the back of nude mice (6 days old, BALB/c, male). On days 2 and 6 post-transplantation, serum phosphate and calcium levels were measured and the short-term effects of recombinant OST311 were examined. CHO ras clone-1 cells were similarly used as a control for this transplantation experiment.

(1) Transplantation of CHO cells

Nude mice (n-6) were each subcutaneously transplanted with 2x10 in a similar manner to the method described in example 11(1)⁷CHO-OST311H cells and 2x10⁷CHO ras clone-1 cells. In addition, equal amounts of PBS (n-6) were similarly implanted subcutaneously. 6 nude mice in each group were housed in a plastic cage, and allowed to freely access the solid food CE-2(CLEA JAPAN, Japan) and tap water. No significant tumor formation was observed 6 days after transplantation.

(2) Determination of serum phosphate and calcium levels at day 2 post cell transplantation

On day 2 after cell transplantation, blood was collected from the eye sockets of mice anesthetized with diethyl ether. Serum was isolated from peripheral blood using a Microtainer (Beckton Dickinson, USA). Serum phosphate levels were measured using P-test Wako (Wako Pure Chemical Industries, Japan), and serum calcium levels were measured using calcium-test Wako (Wako Pure Chemical Industries, Japan). As shown in FIG. 11A, a significant decrease in serum phosphate levels was observed in all members of the CHO-OST311H cell transplant group, as compared with the PBS-administered group and the CHO ras clone-1 cell transplant group. In contrast, serum calcium levels did not change. These results clearly indicate that OST311 only results in a decrease in serum phosphate levels the day after administration.

(3) Determination of serum phosphate and calcium levels at day 6 post cell transplantation

On day 6 after cell transplantation, blood was collected from the heart of mice anesthetized with diethyl ether. Serum phosphate and calcium levels were determined for each group as described above. As shown in FIG. 11B, similar to day 2 after transplantation, a significant decrease in serum phosphate levels was observed in all members of the CHO-OST311H cell transplant group, compared to the PBS-administered group and the CHO ras clone-1 cell transplant group. In contrast, there was only a slight decrease in serum calcium levels in the CHO-OST311H cell transplant group.

EXAMPLE 14 purification of recombinant OST311

CHO-OST311H cells were incubated at 37 ℃ with 5% CO₂And 100% humidity, grown at 225cm²In a culture flask in MEM α medium with 10% FCS. When the cells grew to cover approximately 80% of the area of the bottle, the medium was replaced with 50ml of serum-free medium CHO-S-SFM II (LIFETECHNOLOGY, USA), and after 48 hours, the conditioned medium was collected. The recombinant OST311 was purified by the following method using a total of 1000ml of the conditioned medium thus obtained.

1000ml of the conditioned medium was centrifuged at 16200g for 15 minutes at 4 ℃ to remove suspended cells, and then the supernatant was applied to SP-sepharose FF (Amersham Pharmacia, USA) packed in a glass column (inner diameter 30mmX200mm long). The components flowing out of the column were adsorbed onto Talon Superflow (metal chelating resin, CLONTECH, USA). Non-specific adsorption was removed with a washing buffer containing 50mM sodium phosphate buffer (pH6.6) and 0.3M NaCl, followed by elution with 50mM sodium phosphate buffer (pH6.7) and 0.2M imidazole. The right column of FIG. 12 shows the fractions eluted as detected by Western blotting using an anti-His 6 antibody (INVITROGEN, USA). These fractions contain a partial polypeptide (SEQ ID NO: 8) described in example 9, including amino acid residues 180(Ser) to 251 (Ile). On the other hand, the protein adsorbed on SP-sepharose FF contained in the above conditioned medium was eluted with 50mM sodium phosphate buffer (pH6.7) using a concentration gradient of 0 to 0.7M NaCl. The left column of FIG. 12 shows the fractions eluted as detected by Western blotting using an anti-His 6 antibody (INVITROGEN, USA). These fractions eluted at about 0.3M NaCl contained a partial polypeptide (SEQ ID NO: 4) including amino acid residues 25(Tyr) to 251(Ile) as described in example 9. Furthermore, the middle column of FIG. 12 shows the fractions eluted by Western blotting using the polyclonal antibody (311-114) prepared from the partial peptide of OST311 (SEQ ID NO: 29) as described in example 10. These fractions were eluted at approximately 0.4M NaCl and contained a partial peptide (SEQ ID NO: 6) including amino acid residues 25(Tyr) to 179(Arg) as described in example 9. Thus, the fraction containing three partial peptides of OST311, in particular the peptide of SEQ ID NO: 4 (hereinafter, 311: 25-251), SEQ id no: 6 (hereinafter 311: 25-179) and SEQ ID NO: 8 (hereinafter, 311: 180-251) was purified and separated, and then concentrated using a VIVASPIN column (Sartorius, USA) having an ultrafiltration molecular weight of 10000, followed by replacement with a solvent containing 1ml of 5mM HEPES (pH6.9) and 0.1M NaCl.

Example 15 histological analysis of non-demineralized bone sections

When the CHO-OST311H cell-transplanted mice and tumor-free mice prepared in example 11 were sacrificed, the right leg and tibia were excised, and the knee joint was kept intact. Immediately after the tibial and leg shafts were cut, the legs and tibia were preserved in ice neutral formalin prepared in advance. This produced undemineralized specimens. The method of preparing a demineralized specimen is described below.

Bone tissue was pre-stained with Villanueva bone dye for 3 to 7 days. The tissue was dehydrated through a series of ethanol gradients, followed by acetone replacement of the solvent. After addition of acetone monomer and monomers (After the After acetate monomer and the n monomer wee applied), the tissue samples were embedded in resin. Methyl Methacrylate (MMA) resin was used to embed the samples. The tissue samples were placed in an incubator at about 35 ℃ for complete polymerization. At this point, the tissue was thoroughly embedded in resin by the appropriate addition of MMA. MMA used herein for embedding the sample was prepared by adding and completely dissolving 40g of MMA polymer (Wako Pure Chemical Industries, Japan) to 100ml of MMA monomer (Wako Pure Chemical Industries, Japan), and then adding and dissolving Benzoyl peroxide (Nacalai Tesque, Japan) at a rate of 1g per one part of the solution. A preparation of the tibia is prepared. To enable observation of the cancellous bone of the tibia, the frontal cut was trimmed and a 4um thick frontal cut was prepared with a microtome for hard tissues (RM2065 super cut, Leica). Post-staining was performed with Villanueva-Goldner. The thus-obtained sections were clarified with xylene and then sealed with CLEAR SEAL (MARUTO, Japan) and ONE LIGHT (MARUTO, Japan).

A micrograph of the section is shown in fig. 13. An increase in growth plate width was observed in CHO-OST311H cell transplanted tumor mice compared to the control group. In addition, a significant increase in premorque and a reduced zone of mineralization were observed at the metaphysis. Without evidence of osteitis fibrosis (osteitis fibrosa), bone harvested from tumor mice transplanted with CHO-OST311 cells exhibited typical osteomalacia characteristics.

Example 16 examination of vitamin D metabolism in early stages after transplantation of CHO-OST311H cells

To examine the effect of OST311 on vitamin D metabolism, an experiment of transplanting CHO-OST311H cells into nude mice (6 weeks old, BALB/c, male) was performed in a similar manner to the method described in example 13. Two experimental control groups were established for comparison, including one group similarly transplanted with CHO ras clone-1 cells, and one group given PBS at the same dose as the cell suspension. 6 mice from each group were housed in plastic cages and were given ad libitum access to tap water and solid food CE2(CLEA JAPAN, Japan) containing 1.03% inorganic phosphate and 1.18% calcium. Fluctuations in serum 1, 25-hydroxyvitamin D levels and changes in vitamin D metabolizing enzyme expression were examined on days 1, 2, 3 and 6 post-transplantation.

(1) Determination of serum 1, 25-dihydroxyvitamin D levels

On days 1, 2, 3 and 6 after the cell transplantation, blood was collected from hearts of mice in the PBS-administered group, CHO ras clone-1 cell transplantation group and CHO-OST311H cell transplantation group, which were anesthetized with diethyl ether, respectively, and then serum was isolated using Microtainer (Beckton Dickinson, USA). The same volume of serum collected from each mouse was mixed together to make the total volume of each group 0.25 ml. The level of 1, 25-dihydroxyvitamin D in this was determined using the 1, 25(OH)2D RIA-kit, "TFB" (TFB, Japan). As a result, as shown in Table 6, a significant decrease in the level of 1, 25-dihydroxyvitamin D was observed on day 1 after transplantation in the CHO-OST311H cell transplantation group, as compared with the PBS administration group and the CHO ras clone-1 cell transplantation group. This reduced effect was also observed on days 2, 3 and 6 after transplantation. These results are consistent with a decrease in serum 1, 25-dihydroxyvitamin D levels, which is also a typical clinical finding of tumor-induced osteomalacia.

TABLE 6 serum 1, 25-dihydroxyvitamin D levels in cell-transplanted mice

Post-transplant 1236
Post-transplant 1236	PBS administration group (pmol/L) n-5338164.3164.5273.7 CHO-ras clone-1 cell transplantation 271.9178.3182.9184.6 group (pmol/L) n-6 CHO-OST311 cell transplantation group 46.736.334.549.1 (pmol/L) n-6

(2) Analysis of expression of vitamin D metabolizing enzyme Gene in Kidney

To investigate whether the above-mentioned decreased effect of 1, 25-dihydroxyvitamin D3 was caused by fluctuations in the 25-hydroxyvitamin D-1-alpha-hydroxylase (1. alpha. OHAse) gene or the 25-hydroxyvitamin D-24-hydroxylase (24OHAse) gene, 3 or 4 mice were randomly selected from each of the PBS-administered group, the CHO-ras clone-1 cell-transplanted group and the CHO-OST311H cell-transplanted group on day 3 after transplantation. The kidney was excised, total RNA was prepared according to the procedure described in example 11(7), and then Northern blotting was performed using the probe described in the same example. The results are shown in FIG. 14. The expression level of mRNA of the 1. alpha. OKase gene was significantly reduced in the CHO-OST311H cell transplantation group, as compared with the PBS administration group and the CHO-ras clone-1 cell transplantation group. This result suggests the possibility that OST311 inhibits the expression of this gene directly or indirectly, thereby inhibiting the biosynthesis of serum 1, 25-dihydroxyvitamin D. On the other hand, the mRNA expression level of the 24OHase gene was significantly increased in the CHO-OST311H cell transplantation group, as compared to the PBS-administered group and the CHO-ras clone-1 cell transplantation group. This result suggests the possibility that OST311 directly or indirectly enhances the expression of this gene, thereby promoting the inactivation of serum 1, 25-dihydroxyvitamin D.

In example 11(8), no significant difference in serum 1, 25-dihydroxyvitamin D levels was observed at days 44 and 46 after transplantation, compared to the control group. Another result was different from the present example in that the expression level of 1. alpha. OHAse mRNA tended to increase. At least one possible explanation is that this difference is due to the effects of serum parathyroid hormone described in example 17.

Example 17 early examination of serum parathyroid hormone levels after CHO-OST311H cell transplantation

On days 1, 2, 3, 6 and 45 after transplantation of CHO cells as described in examples 11, 13 and 16, collected aliquots of mouse serum were mixed together in a total volume of 0.15 ml. Serum parathyroid hormone levels were then determined using the Rat PTH IRMA kit (Nihon Medi-Physics, Japan) according to the attached manufacturer's instructions. As shown in Table 7, a significant increase in serum parathyroid hormone levels was observed in the CHO-OST311 transplant group, with the difference being evident at day 45 post-transplant.

TABLE 7 Parathyroid hormone levels in cell-transplanted mice

After transplantation 1 2 3 6 45
After transplantation 1 2 3 6 45	PBS administration group (pg/ml) n-545.223.828.219.7¹41.9CHO-ras clone-1 cell transfer 15.826.615.713.8²40.4 Implantation group (pg/ml) n-6 CHO-OST311 cell transplantation group 13.820.64457.8³211.7(pg/ml)n＝6

¹Measurement when tumor-free mice (n-6) were used.²n＝10，³n＝12。

Example 18 experiment of administration of recombinant OST311H full-Length protein produced by CHO in Normal mice

To investigate the effect of CHO-produced recombinant OST311H full-length protein on normal mice (BALB/C, male, 6 weeks old), a polypeptide having a histidine tag at the C-terminus, which polypeptide includes the 25 th amino acid residue, Tyr to residue 251Ile (SEQ ID NO: 4), was partially purified by the purification method described in example 14 (1). The purified fractions were administered intraperitoneally at 0.1ml each time to normal mice. From the fluorescence intensity obtained by Western blotting, the purified fraction was estimated to contain about 0.15 to 0.75ug of recombinant OST 311. Similar to example 14, 0.1ml of a solvent (5mM HEPES buffer/0.1M NaCl, pH7.0) was intraperitoneally administered to the control group. The OST311 administration group and the control group included 5 mice, respectively. 5 mice of each group were housed in plastic cages and allowed ad libitum access to tap water and solid food CE2(CLEA JAPAN, Japan) containing 1.03% inorganic phosphate and 1.18% calcium.

[ experiment 1]

The outline of the experiment is shown in FIG. 15A. Additional doses were given at 5, 10, 23, 28 and 33 hours after the first intraperitoneal dose. Thus, a total of 6 intraperitoneal administrations were performed. Subsequently, blood was collected from the orbital fossa at 36, 47 and 71 hours after the first intraperitoneal administration using a glass capillary, and then serum was separated using a Microtainer (Beckton Dickinson, USA).

Phosphate and calcium levels in the serum thus obtained were determined using P-test Wako or calcium-test Wako (Wako Pure chemical industries, Japan) according to the attached manufacturer's instructions. As shown in FIG. 15B, the effect of significant decrease in serum phosphate level was observed in the OST 311-administered group at 36 hours after the first administration (t-test)^**p＜0.001，^*p is less than 0.01). Furthermore, this effect was maintained for 11 hours after this time (47 hours after the first dose). On the other hand, such activity disappeared 71 hours after the first administration (38 hours after the last administration). In addition, no significant change in serum calcium levels was found at any time (fig. 15C).

[ experiment 2]

The outline of the experiment is shown in FIG. 16A. Additional doses were administered 5 and 11 hours after the first intraperitoneal dose. Thus, a total of 3 intraperitoneal administrations were performed. Subsequently, blood was collected from the orbital fossa by a glass capillary at 13 and 24 hours after the first intraperitoneal administration, and then serum was separated by Microtainer (beckton dickinson, USA). The phosphate and calcium levels in the serum thus obtained were determined using either P-test Wako or calcium-test Wako according to the attached manufacturer's instructions. As shown in FIG. 16B, a significant decrease in serum phosphate levels was observed in the OST 311-administered group 13 hours after the first administration (t-test)^**p＜0.05，^*p is less than 0.01). Furthermore, this effect was maintained for 11 hours after this time. In addition, no significant change in serum calcium levels was found at any time (fig. 16C).

The results of experiments 1 and 2 revealed that intraperitoneal administration of the full-length fraction of recombinant CHO-produced OST311 protein to normal mice induced hypophosphatemia, a decrease in serum phosphate levels having been observed at 13 hours after the first administration, and this activity was maintained for 11 hours after the cessation of administration.

Example 19 introduction of mutations into OST311

As shown in example 9, a part of the recombinant OST311 produced by CHO-OST311H cells was cleaved during its secretion into a polypeptide having a sequence from Tyr 25 to Arg 179 (SEQ ID NO: 6) and a polypeptide having a sequence from Ser 180 to Ile 251 (SEQ ID NO: 8).

The cleavage may be due to a protease recognizing a motif comprising the RXR sequence or the RRXXR sequence located just before Ser at amino acid residue 180. When a full-length recombinant is administered to a living organism, the recombinant is considered to be likely to undergo such cleavage activity-like degradation. Thus, OST311RQ gene encoding a sequence in which Arg at position 176 and Arg at position 179 are substituted with Gln was prepared for introduction of mutation.

(1) Preparation of OST311/pCAGGS plasmid

PCR was carried out with LA Taq polymerase (TAKARA SHUZO, Japan) using OST311H/pcDNA3.1 plasmid as a template and 311F1EcoRI (SEQ ID NO: 22) and 311LNot (SEQ ID NO: 44) as primers. After maintaining the temperature at 94 ℃ for 1 minute, 25 cycles of reaction were carried out, each cycle consisting of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute. After completion of the reaction, the PCR product was degraded to blunt ends with T4 DNA polymerase (Roche, Swiss), and then phenol-chloroform treatment was performed to inactivate the enzyme. The DNA was precipitated with ethanol and then phosphorylated at the ends with polynucleotide kinase (Roche, Swiss). The target DNA fragment was separated by 0.8% agarose gel electrophoresis and then recovered by Gene Clean II (BIO101, USA). The plasmid vector pCAGGS (Niwa H et al, Gene 199112, 15; 108(2)) was digested with EcoRI and then blunt-ended with the Klenow fragment (Roche, Swiss). The DNA was subsequently end dephosphorylated with bovine small intestine alkaline phosphatase (TAKARASHUZO, Japan). The target DNA fragment was separated by 0.8% agarose gel electrophoresis and then recovered by Gene Clean II (BIO101, USA). The OST311cDNA thus obtained was ligated to a previously digested pCAGGS plasmid using a DNA ligation kit (2 nd edition) (TAKARA SHUZO, Japan) according to the instructions of the appended manufacturer. The product was introduced into E.coli DH 5. alpha. for cloning, thus obtaining the relevant plasmid. This plasmid was used to prepare the OST311RQH gene.

311LNot：ATAAGAATGCGGCCGCTCAGATGAACTTGGCGAA(SEQ IDNO：44)

(2) Preparation of OST311RQH Gene

The following primers were synthesized.

OST311ME1：ATGAATTCCACCATGTTGGGGGCCCGCCTCAGG(SEQ IDNO：45)

OST311HNt：ATGCGGCCGCCTAATGATGATGATGATGATGGATGAACTTGGCGAAGGG(SEQ ID NO：46)

OST311RQF：ATACCACGGCAGCACACCCAGAGCGCCGAG(SEQ ID NO：47)

OST311RQR：CTCGGCGCTCTGGGTGTGCTGCCGTGGTAT(SEQ ID NO：48)

OST311ME1 is a forward primer containing a portion of the headleucine of OST311, OST311HNt is a reverse primer with 6 histidines added to the 3' end of OST311, OST311RQF and OST311RQR are forward and reverse primers for introducing mutations, replacing the guanines at positions 527 and 536 (corresponding to guanines 659 and 668 of SEQ ID NO: 1), respectively, of the coding region of OST311cDNA with adenine, thereby replacing the arginines of amino acids 176 and 179 with glutamine. Two reaction solutions were prepared at 20ul each using pfu DNA polymerase (Promega, USA) according to the attached manufacturer's instructions. On the other hand, OST311ME1 and OST311RQR were used as primers at a final concentration of 0.2uM, and 10ng of the OST311 expression vector described in example 6(1) was used as a template. After maintaining the temperature at 94 ℃ for 1 minute, 25 PCR reactions were performed, each cycle consisting of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 1 minute. Further, OST311RQF and OST311HNt at a final concentration of 0.2uM were used as primers, and 10ng of OST311/pCAGGS plasmid was used as a template. After maintaining the temperature at 94 ℃ for 1 minute, 35 PCR reactions were performed, each cycle consisting of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 1 minute. The above two reaction products were diluted 10-fold, respectively, and then 1ul of each solution was added to 50ul of a reaction solution prepared using LA Taq polymerase (TAKARA SHUZO, Japan) according to the instructions of the attached manufacturer. After maintaining the temperature at 94 ℃ for 1 minute, 25 PCR cycles of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 1 minute were carried out using LA Taq polymerase with OST311ME1 and OST311HNt as primers at a final concentration of 0.2 uM. After completion of the PCR reaction, the solution was maintained at 72 ℃ for 7 minutes. The reaction product thus obtained was subjected to phenol/chloroform treatment, deproteinization, ethanol precipitation, and then digestion with EcoRI and NotI. A DNA fragment of about 800bp was separated by 2% agarose gel electrophoresis and then recovered by Gene Clean II (BIO101, USA). The resulting DNA fragment was inserted into EcoRI and NotI sites of vector IRES-EGFP-pEAK8 to obtain OST311RQH/IRES-EGFP/pEAK8 plasmid, wherein the IRES-EGFP-pEAK8 vector was prepared by ligating an Internal Ribosome Entry Site (IRES) and Enhanced Green Fluorescent Protein (EGFP) to plasmid pEAK8(EdgeBioSystems, USA). Plasmid DNA was prepared according to a standard method, and then the nucleotide sequence was determined using ABI3700 fluorescent DNA sequencer (PE applied systems, USA), which confirmed that the sequence encodes a polypeptide into which the mutations of R176Q and R179Q were introduced, and a histidine tag was added at the C-terminus. The polypeptide encoded by this gene is hereinafter referred to as OST311 RQH.

(3) Isolation of CHO cells stably expressing OST311RQH

The OST311RQH/IRES-EGFP/pEAK8 plasmid was introduced into CHO-ras clone-1 cells using Transfectam (Promega, USA) according to the instructions of the attached manufacturer. Drug-resistant cells were selected on MEM α medium containing 5ug/ml puromycin and 10% FCS. Then, cells with high fluorescence intensity of GFP (green fluorescent protein) were panned by FACS vantage (Beckton Dickinson, USA), followed by cloning. When the clonal cells were confluent, the medium was replaced with serum-free DF medium (DMEM/F-12), and then conditioned medium was collected 2 days after medium replacement. 50ul of the collected conditioned medium was adsorbed onto Immobilon P filters (Millipore, USA) using a 96-well variable filter system (Lifetechiental, USA). The prepared filters were washed with TBS and TTBS and then blocked with Blockace (Daiichi pharmaceutical, Japan) at room temperature for 1 hour. After blocking, the membrane was reacted with a 5000-fold dilution of HRP-labeled anti-His 6 monoclonal antibody (Invitrogen, USA) with Blockace for 1 hour. After the reaction, the filter was washed with TTBS and TBS, and then the signal was detected with ECL (Amersham Pharmacia, USA) according to the attached manufacturer's instructions. Based on the signal intensity, the high expression clone CHO-OST311RQH was selected.

(4) Preparation of conditioned Medium for OST311RQH pEAK Rapid cells

pEAK rapid cells (EdgeBioSystems, USA) were seeded in 20 flasks of tissue culture (225cm2, CORNING, USA). The cells were transfected with 0.48mg of OST311RQH/IRES-GFP/pEAK8 plasmid by the calcium phosphate method according to the manufacturer's instructions attached to the pEAK System (EdgeBioSystems, USA). The cells were left for 4 hours. Next, the medium in each flask was replaced with 50ml of serum-free MEM α medium, the cells were cultured at 37 ℃ for 2 days, and then the conditioned medium was collected.

(5) Demonstration of recombinant OST311RQH expression

From the conditioned medium of the two CHO-OST311RQH cell clones described above and of the transient expression of pEAK rapid cells, 10ul of each was subjected to Western blotting in the manner described in example 6(3) to examine the culture supernatant for the presence of recombinant OST311 RQH. An anti-His (C-terminal) antibody (Invitrogen, USA) was used as a detection antibody. As shown in FIG. 17, a strong signal at the same position as the band of about 32kDa described in (3) of example 6 was observed in all the culture supernatants. Also, no signal of about 10kDa was observed for Western blot for all conditioned media. From these results, it can be concluded that the introduction of the mutations R176Q and R179Q results in the inhibition or attenuation of the cleavage of the polypeptide that is expected to occur at these positions, and therefore the ratio of polypeptides having a sequence from amino acids 180Ser to 251Ile is significantly reduced.

Example 20 recombinant OST311RQH administration experiment in Normal mice

From 500ml of the culture supernatant prepared in example 19(5), purified fractions containing about 2.8ug/ml of recombinant OST311RQH protein were obtained according to the method described in example 14 (1). The purified fractions were administered intraperitoneally at 0.1 ml/time consecutively to normal mice (BALB/c, male, 6 weeks old) and then serum phosphate, calcium and 1, 25-dihydroxyvitamin D levels were measured. For the control group, 0.1ml of the vehicle (5mM HEPES buffer/0.1M NaCl pH7.0) was similarly administered intraperitoneally, respectively. The OST311 RQH-administered group and the control group each comprised 6 mice. 6 mice of each group were housed in a plastic cage, allowing ad libitum access to tap water and solid food CE2(CLEA JAPAN, Japan) containing 1.03% inorganic phosphate and 1.18% calcium.

The experimental protocol is shown in figure 18A. Additional doses were administered at 5, 10, 24, 29 and 34 hours after the first dose. Thus, a total of 6 consecutive administrations were given. In the above procedure, blood was collected from the orbital fossa of the diethyl ether-anesthetized mouse by a glass capillary at 24 hours after the first dose (before the 4 th dose), and from the heart at 48 hours after the first dose.

(1) Measurement of serum phosphate and calcium levels

Sera were collected 24 and 48 hours after the first dose and serum phosphate levels were measured by the method described in example 14 (3). As a result, the OST311 RQH-administered group showed significant hypophosphatemia at any time of blood sampling, as shown in FIG. 18B (t-test)^**p＜0.01，^*p < 0.05). In contrast, there was no significant fluctuation in serum calcium levels (fig. 18C).

(2) Measurement of serum 1, 25-dihydroxyvitamin D levels

Equal volumes of serum from each mouse were pooled together 48 hours after the first dose. Then, the serum 1, 25-dihydroxyvitamin D level was measured by the method described in example 16 (1). The control group showed 244.7pmol/L, while the OST311 RQH-administered group showed a significant drop, i.e., 24.6 pmol/L.

Example 21 CHO-OST311RQH cell transplantation experiment

An experiment was carried out in analogy to the procedure described in example 13, in which cells stably expressing OST311RQH as described in example 19(3) were transplanted into nude mice (7 weeks old, BALB/c-nude, male, n ═ 8). CHO ras clone-1 cells were similarly transplanted as a control group (n ═ 6). Each group of nude mice was housed in a plastic cage and allowed to take tap water and solid food CE2(CLEA JAPAN, JAPAN) containing 1.03% inorganic phosphate and 1.18% calcium ad libitum.

On day 2 after cell transplantation, blood was collected from the eye socket using a glass capillary, and then serum phosphate and calcium levels were measured by a similar method as described in example 14 (3). As shown in FIG. 19A, a significant decrease in serum phosphate levels was observed in the CHO-OST311RQH cell transplant group (t-test)^*p < 0.001), while serum calcium levels did not change significantly (fig. 19B).

EXAMPLE 22 preparation of polyclonal antibody against OST311 partial peptide (2)

4 partial OST311 peptides were prepared in a similar manner as described in example 10. Using these peptides as antigens, rabbits were immunized and then Western blotting was performed using the obtained antiserum according to the method described in example 6(3), whereby recombinant OST311H was detected in the conditioned medium of CHO-OST311H cells. The antibody reaction was stirred overnight at 4 ℃ in a solution prepared by diluting the antiserum to each peptide 250-fold with TTBS. After washing, alkaline phosphatase-labeled goat anti-rabbit antibody (DAKO, Denmark) was added to the solution for binding, and recombinant OST311 was detected with alkaline phosphatase staining kit (BIO-RAD, USA) (fig. 20).

Partial peptide

311-148：GMNPPPYSQFLSRRNEC(SEQ ID NO：49)

311-170：CNTPIPRRHTR(SEQ ID NO：50)

311-180：SAEDDSERDPLNVLKC(SEQ ID NO：51)

311-210：LPSAEDNSPMASDC(SEQ ID NO：52)

Example 23 construction of ELISA System for detecting OST311 protein

(1) Purification of antibodies from Rabbit antiserum raised against partial peptide OST311

The EcoRO-Pac degradable chromatography column (BIO-RAD, USA) was packed with 3ml of protein Sepharose4FF (Amersham Pharmacia, USA) slurry, and then washed with 10ml of 0.1M glycine hydrochloride buffer (pH3.3) and 20ml of PBS. Two rabbit antisera as described in example 10 and 900ul each of the 4 rabbit antisera as described in example 22 were added to adsorb the antibody component onto the resin. The column was washed with 9ml of PBS to remove contaminants, and then 1ml of 0.1M glycine hydrochloride buffer solution (pH3.3) was added to each column to obtain IgG eluate fractions. For elution, 10ul of a neutralization buffer (1M Tris) was added to each fraction as needed to neutralize the solution. The light absorption at 280nm was measured to determine the concentration of the antibody contained in the eluted fractions (light absorption calculated as: 1.34(mg/ml)^-1.(cm)^-1). Then, some fractions were loaded together onto a NAP25 column, the solvent being replaced with 50mM sodium bicarbonate solution. As a result, 5 to 15mg of antibodies (these polyclonal antibodies are hereinafter referred to as 311-48 antibody, 311-114 antibody, 311-148 antibody, 311-170 antibody, 311-180 antibody and 311-210 antibody, respectively) were obtained from each peptide antiserum.

(2) Biotinylation of anti-OST 311IgG

The above 6 polyclonal antibodies against OST311 peptide were diluted to 1mg/ml in 50mM sodium bicarbonate solution. Then, 1mg each of the antibodies dissolved in 10. mu.l of dimethylformamide was mixed with a solution (1.82ug/ml) of Biotin-AC5-Osu (Japan, Dojindo) by inversion at 4 ℃ for 2 hours to mix well. Subsequently, unreacted Biotin-AC5-Osu in the mixed solution was removed by NAP10 column and the solvent was replaced with PBS to obtain 6 kinds of biotinylated polyclonal antibodies against OST311 peptide.

(3) Detection of OST311 in conditioned Medium expressing OST311 cells by Sandwich ELISA Using Rabbit polyclonal antibody against OST peptide

A sandwich ELISA system was constructed by combining 6 polyclonal antibodies against OST311 peptide for immobilization and the 6 biotinylated antibodies for detection described above. Thus, the detection of OST311 protein in the conditioned medium of OST 311-expressing cells was examined.

The 6 anti-OST 311 peptide polyclonal antibodies for immobilization obtained by purifying protein A as described above were diluted to 10ug/ml in 50mM sodium bicarbonate solution. 50ul of each dilution was added to each well of a 96-well ELISA plate Maxisorp (Nunc, USA), and then left at 37 ℃ for 1 hour to immobilize IgG. Next, the reaction was aspirated, and 50ul of Superlock blocking buffer in TBS was added to each well and blocked for 10 min at room temperature. After the solution was aspirated, the OST311RQH peak fast culture supernatant described in example 19(5) or MEM α medium as a control was added to 50. mu.l per well, and then allowed to stand at room temperature for 1 hour to bind to the immobilized antibody. After the antibody reaction, the solution was washed 3 times with TTBS, 50ul each of the above 6 biotinylated anti-OST 311 antibodies (311-48, 311-114, 311-148, 311-170, 311-180 and 311-210) containing 10% of Blockace (Dainippon pharmaceutical, Japan) diluted to 10ug/ml in TTBS were added to each well, followed by standing at room temperature for 30 minutes for the second antibody reaction. Each well was washed 3 times with TTBS, then 50ul of HRP-labeled streptavidin (DAKO, Denmark) containing 10% Blockace diluted 10000-fold with TTBS was added, and then allowed to stand at room temperature for 30 minutes to bind to the biotinylated antibody. Each well was then washed 3 times with TTBS, 50. mu.l of tetramethyl benzidine was added to each well, a peroxidase chromogenic substrate (DAKO, Denmark) was added, and then color formation was performed at room temperature for 5 minutes. The reaction was then stopped by adding 50. mu.l of 0.5M sulfonic acid solution to each well. The measurement was carried out with a light absorption measurement system MTP300(CORONA ELECTRIC, Japan) suitable for 96-well plates, with the light absorption at 450nm divided by the light absorption at 570 nm. When MEM α alone was added as a control, the values obtained at 450nm/570nm in each case were 0.22 or less. In contrast, as shown in FIG. 21A, when the 311-48 immobilized antibody was used for detecting binding with the 311-180 antibody, or when the 311-180 immobilized antibody was used for detecting binding with the 311-148 immobilized antibody, the OST311RQH in the conditioned medium was detected to be significantly higher than that in the control group. Furthermore, when the 311-48 antibody was immobilized and such a combination was detected using the 311-148 antibody, it was concluded that not only the full-length polypeptide but also an N-terminal portion polypeptide fragment could be detected, since the antigenic sites of both antibodies contained the N-terminal portion peptide (SEQ ID NO: 6) after the cleavage site of the OST311 protein described in example 9. In contrast, when such a combination was detected using 311-210 antibody and 311-80 antibody, it was concluded that not only the full-length polypeptide but also the C-terminal portion peptide (SEQ ID NO: 8) following the cleavage site described in example 9 could be detected. Thus, the multiple uses of these combinations enable the measurement of the absolute amounts and ratios of the full length polypeptide and partial polypeptide of OST311 in a sample, such as a biological sample.

(4) Quantification of recombinant OST311 protein concentration by Sandwich ELISA Using Rabbit polyclonal antibody against OST311 peptide

In the above ELISA system, the detection of purified recombinant OST311 was examined using a combination of 311-48 antibody or 311-180 antibody as the immobilized antibody and 311-148 antibody as the detection antibody, wherein OST311 includes a series of dilutions of 1, 0.67, 0.33, 0.1, 0.067, 0.033 and 0.01. mu.g/ml. As shown in FIG. 21B, good linearity can be obtained in the range of 0.1 to 1. mu.g/ml (311-48: R)²＝0.990，311-180：R²0.996). The results revealed that at least recombinant OST311 can be detected in this concentration range.

EXAMPLE 24 examination of Effect of Once administration of recombinant OST311 protein

To examine the short-term effect of CHO-produced recombinant OST311H full-length protein on normal mice (BALB/c, male, 6 weeks old), purified recombinant OST311H full-length protein was administered once via the tail vein at 5.0ug/0.1ml per mouse. For the control group, 0.1ml of vehicle (PBS) was administered to each mouse via the tail vein. At 1, 3 and 8 hours after administration, blood was collected from the heart and dissected to measure serum phosphate, calcium and vitamin D levels, and then the expression level of sodium-phosphate cotransporter protein on the renal proximal tubule was analyzed. The OST 311-administered group and the control group included 6 mice, respectively. 6 mice of each group were closed and allowed ad libitum access to tap water and solid food CE2(CLEA JAPAN, Japan) containing 1.03% inorganic phosphate and 1.18% calcium.

(1) Changes in serum phosphate levels over time

As shown in table 8, no significant changes in serum phosphate levels were observed 1 and 3 hours after one administration of OST311 protein, and a significant drop was observed 8 hours after administration. This result illustrates that the effect of OST311 takes 3 to 8 hours to reduce serum phosphate levels. On the other hand, no change in serum calcium levels was observed at any time.

TABLE 8 serum phosphate levels

Time of day	1	3	8
Time of day	1	3	8	Vehicle administration group (mg/dL)	9.82±0.61	9.99±0.20	9.55±0.29
OST311 administration group (mg/dL)	9.61±0.51	9.96±0.39	7.82±0.27	Vehicle administration group (mg/dL)	9.82±0.61	9.99±0.20	9.55±0.29
OST311 administration group (mg/dL)	9.61±0.51	9.96±0.39	7.82±0.27	t-test	P＞0.5	P＞0.5	P＜0.005

(2) Expression of sodium-phosphate cotransporter protein in renal proximal tubules

Kidneys collected at 1, 3 and 8 hours after administration were mixed together for each group, and brush-insulating membrane (BBM) of proximal tubule was prepared according to the method described in example 11 (6). The resulting BBM was analyzed by Western blot for the ratio of sodium-phosphate in combination with carrier protein (NaPi 7). As shown in fig. 22A, the expression level of NaPi7 was comparable to that of the vector-administered group 1 and 3 hours after administration, and the level of NaPi7 was significantly lower in the OST 311-administered group than in the vector-administered group 8 hours after administration. Meanwhile, in order to verify whether reduction of NaPi7 protein is associated with RNA transcription regulation, total RNA was prepared from the excised kidney of each mouse according to the procedure described in example 11(7), and Western blotting was performed using the probe described in the same example. Thus, as shown in fig. 22B, the mRNA levels of NaPi7 were comparable to the vehicle-administered group 1 and 3 hours after administration, but the NaPi7 was significantly reduced in the OST 311-administered group compared to the vehicle-administered group 8 hours after administration. The above results clearly indicate that the decrease in serum phosphate levels is due to the direct or indirect effect of recombinant OST311 protein, which down-regulates sodium-phosphate cotransporter protein on the renal tubules, and that inhibition of NaPi7 at the mRNA transcription level is at least one factor in the down-regulation of protein levels.

(3) Serum 1, 25-dihydroxyvitamin D3 levels over time

Serum 1, 25-dihydroxyvitamin D3 levels were measured at 1, 3 and 8 hours post-administration by the method described in example 16 (1). As shown in fig. 23, in the OST 311-administered group, a significant decrease in serum 1, 25-dihydroxyvitamin D3 level was observed 3 hours after administration, and a further decrease was observed 8 hours after administration.

(4) Expression Change of vitamin D metabolizing enzyme Gene

To elucidate whether the decrease in serum 1, 25-dihydroxyvitamin D3 levels was due to fluctuations in the expression of the 25-hydroxyvitamin D-1-alpha-hydroxylase (1. alpha. OHAse) or 25-hydroxyvitamin D-24-hydroxylase (24OHAse) gene in the kidney, total RNA was prepared from the kidney at 1, 3, and 8 hours after administration according to the procedure described in example 11(7), and then Western blotting was performed using the probe described in this example. As shown in fig. 24, a decrease in mRNA level of the 1 α OHase gene and an increase in mRNA level of the 24OHase gene were observed at 1 hour after the administration. This trend appears more pronounced at 8 hours post-administration. In FIG. 24, the vector represents a solvent for recombinant OST311 protein, which contains 20mM phosphate buffer (pH6.7) and 0.3M NaCl.

These results show that OST311 reduces serum 1, 25-dihydroxyvitamin D3 levels by modulating the expression of the 25-hydroxyvitamin D-1-alpha-hydroxylase (1 alpha OHase) or 25-hydroxyvitamin D-24-hydroxylase (24OHase) genes in the kidney.

Example 25 examination of the Activity of C-terminally deleted OST311

(1) Construction of an OST311 expression System lacking the C-terminal portion

The following primers were synthesized.

OST311R693 ATGCGGCCGCTATCGACCGCCCCTGACCACCCC(SEQ ID NO：53)

OST311R633 ATGCGGCCGCTACGGGAGCTCCTGTGAACAGGA(SEQ ID NO：54)

OST311R618 ATGCGGCCGCTCAACAGGAGGCCGGGGCCGGGGT(SEQ ID NO：55)

OST311R603 ATGCGGCCGCTCACGGGGTCATCCGGGCCCGGGG(SEQ ID NO：56)

OST311R693, OST311R633, OST311R618 and OST311R603 are reverse primers for deleting 20, 40, 45 and 50 amino acid residues from the 3' end of OST311, respectively, and introducing a stop codon and a NotI recognition sequence. Each of these reverse primers was combined with the forward primer OST311ME1(SEQ ID NO: 45) containing the initiation leucine of OST311 and the recognition sequence for EcoRI described in example 19 to a final concentration of 0.2 uM. Using these primers, Pyrobest DNA polymerase (TAKARA SHUZO, Japan) and 100ng of the plasmid DNA OST311RQH/IRES-EGFP/pEAK8 described in example 19(2) as a template, 25 PCR reactions were carried out after maintaining the temperature at 94 ℃ for 1 minute. Each cycle consisted of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute. The resulting reaction product was phenol/chloroform treated, deproteinized, and then ethanol precipitated. The reaction product was then digested with EcoRI and NotI, subjected to 2% agarose gel electrophoresis to separate each DNA fragment and recovered with Gene CleanII (BIO101, USA). The resulting DNA fragment was ligated to pEAK8 vector (EdgeBioSystems, USA) digested with EcoRI and NotI to obtain pPKOST 311-. DELTA.C 20, -DELTA.C 40, -DELTA.C 45, -DELTA.C 50 plasmids. Plasmid DNA was prepared by a standard method, and then the nucleotide sequence was determined by an ABI3700 fluorescent DNA sequencer (PE Applied Biosystems, USA), thereby confirming that base pairs have been deleted from the 3' end of OST311RQH gene as required.

(2) Isolation of CHO cells stably expressing recombinants

pPKOST 311-. DELTA.C 20, - Δ C40, - Δ C45, - Δ C50 plasmid DNA was introduced into CHO ras clone-1 cells, respectively, using Transfectam (Promega, USA) according to the instructions of the appended manufacturer. CHO-OST311RQ- Δ C20, - Δ C40, - Δ C45 and- Δ C50 cells showing drug resistance on MEM α medium containing 5ug/ml puromycin and 10% FCS were obtained. These cells were seeded into 24-well culture plates, respectively, and then cultured to confluency on MEM α medium containing 5ug/ml puromycin and 10% FCS. Subsequently, the medium was replaced with serum-free DF (DMEM/F-12) medium. After 3 days, the conditioned medium was collected. The conditioned medium was subjected to Western blotting using the OST 311-specific polyclonal antibody 311-148 or 311-180 described in example 22 to confirm that the relevant protein was expressed at the position corresponding to the predicted molecular weight.

(3) Transplantation test of CHO cells expressing C-terminal-deleted OST311

CHO cells expressing the above OST311 deleted for 20, 40, 45 and 50 residues were each subperitoneally transplanted into nude mice (6 weeks old, BALB/c-nude, male, 6 per group) in a similar manner to the method described in example 13. As a control group, CHO cells expressing full-length OST311RQH and CHO ras clone-1 cells (n ═ 6) were transplanted intraperitoneally, respectively. Mice in each group were housed in plastic cages and allowed ad libitum access to tap water and solid food CE2 (clearjapan, Japan).

On day 3 post-transplantation, blood was collected from the heart, and then serum phosphate, calcium, and 1, 25-dihydroxyvitamin D3 levels were measured by a similar method as described in example 20. As shown in fig. 25, in all CHO-OST311 RQ-ac 20, -ac 40, -ac 45 and-ac 50 cell transplant groups, a significant decrease in serum phosphate levels was observed, comparable to the group into which cells expressing full-length OST311RQH were transplanted (t-test,^**p < 0.001). Also, significant reductions in serum 1, 25-dihydroxyvitamin D3 levels were observed in the CHO-OST311RQ- Δ C20, - Δ C40, - Δ C45 and- Δ C50 cell transplant groups (full length: 3.1%, Δ C40: 9.4%, Δ C45: 10.0% and Δ C50: 68.1% when the average serum level in the CHO ras clone-1 transplant group was defined as 100%). These results clearly show that even if 50 amino acids are deleted from the C-terminus of OST311 protein, its serum phosphate-lowering activity or serum 1, 25-dihydroxyvitamin D3 level-lowering activity is retained.

Example 26 Activity assay of N-terminal deletion of OST311

(1) Construction of OST311 expression System lacking the N-terminal 9 amino acid residues

The following oligo-DNA was synthesized.

OST311SGFW：

aattccaccATGTTGGGGGCCCGCCTCAGGCTCTGGGTCTGTGCTTGTGCAGCGTCTGC

AGCATGAGCGTCCTgcatGC(SEQ ID NO：57)

OST311SGRV：

aattGCatgcAGGACGCTCATGCTGCAGACGCTGCACAAGGCACAGACCCAGAGCCTG

AGGCGGGCCCCCAACATgg^tgg(SEQ ID NO：58)

OST311SGFW is a peptide comprising from the start leucine of OST311 to SEQ ID NO: 2, which contains the gene sequence coding for the signal peptide and has an EcoRI recognition sequence at the 5' end. OST311SGRV is the complementary strand of OST311SGFW and contains an EcoRI recognition sequence at the 5' end. Furthermore, recognition sites for the restriction enzyme SphI were introduced in the 3 'direction of OST311SGFW and in the 5' direction of OST311 SGRV. The introduction of the SphI recognition site replaced the 23 rd amino acid Arg in the signal peptide sequence with His. The above oligo-DNA was annealed according to a standard method to obtain a double-stranded DNA fragment containing EcoRI recognition sequences at both ends, a SphI recognition sequence at a position corresponding to the 23 rd amino acid residue of the signal peptide, a full-length signal peptide coding for leucine starting from OST311 and a modified residue. The resulting DNA fragment was inserted into pEAK8 vector (EdgeBioSystems, USA). Then, plasmid DNA in which the EF1 promoter and the above DNA fragment were present in the vector in the forward direction was selected to obtain plasmid pPKFGSG.

Next, the following primers were synthesized.

OST311dN9：ATATGCATGCCTCCAGCTGGGGTGGCCTGATCCAC(SEQ ID NO：59).

OST311dN9 is a forward primer designed to contain a SphI recognition site at its 5' end, SEQ ID NO: 2 followed by the 24 th amino acid residue Ala from SEQ ID NO: 2 at amino acid residue 34, Ser. Using this primer in combination with a reverse primer OST311HNt (example 19, SEQ ID NO: 46) having a NotI recognition sequence with the addition of 6 histidine residues at the C-terminus followed by a stop codon, PCR amplification was carried out in the same manner as described in example 25(1) using OST311RQH/IRES-EGFP/pEAK8 plasmid DNA described in example 19(2) as a template. The resulting PCR product was digested with SphI and NotI and then inserted into the SphI-and NotI-digested plasmid vector pPKFGSG described above according to standard methods.

The nucleotide sequence of the resulting plasmid OST 311. DELTA.N 9-pPKFGSG was determined using an ABI3700 fluorescent DNA sequencer (PE Applied Biosystems, USA), whereby it was confirmed that the inserted gene sequence contained a signal peptide from the initiator leucine to the 24 th amino acid Ala of the OST311 RXH gene (in which the 23 rd amino acid Arg was substituted by His), a gene sequence corresponding to 9 amino acid residues from the 25 th amino acid Tyr to the 33 rd amino acid Gly was deleted, and the entire sequence from the 34 th amino acid Ser to the histidine tag containing the termination codon.

(2) Isolation of CHO cells stably expressing recombinant OST 311. DELTA.N 9

OST 311. DELTA.N 9-pPKFGSG plasmid DNA was introduced into CHO ras clone-1 cells using Transfectam (Promega, USA) according to the attached manufacturer's instructions, and CHO-OST311 RQ-. DELTA.N 9 cells showing drug resistance on MEM. alpha. medium containing 5ug/ml puromycin and 10% FCS were obtained. Conditioned media was collected from the resulting cells in the manner described in example 25. The OST 311-specific polyclonal antibody 311-148 described in example 22 was then used or the fragment was generated using the sequence shown in SEQ ID NO: 2 from 237Gly to 251Ile in the sequence of. Thus, it was confirmed that the protein at the position corresponding to the expected molecular weight was expressed. These results reveal the OST311 signal peptide, which is shown in SEQ ID NO: 2 by His, it is possible to ensure secretion of the OST311 recombinant protein, and even if at least a part of amino acids 25Tyr to 33 Gly is deleted, the recombinant protein can be stably present in the medium to some extent.

(3) Transplantation test of cells expressing OST311 having N-terminal deletion of 9 amino acids

The CHO-OST311RQ- Δ 9 cells above were subperitoneally transplanted into nude mice (8 weeks old, BALB/c-nude, male, 6 per group) in a similar manner to the method described in example 13. As a control group, CHO cells and CHO ras clone-1 cells (n ═ 6) expressing full-length OST311RQH were implanted similarly in the peritoneum, respectively. Mice from each group were housed in plastic cages, allowing ad libitum access to tap water and solid food CE2(CLEA JAPAN, JAPAN).

4 days after cell transplantation, blood was collected from the orbital using a glass capillary, and then serum phosphate level was measured in a similar manner as described in example 20. A significant decrease in serum phosphate levels was observed in the CHO-OST311RQ Δ 9 cell transplant group (CHO ras clone-1 group: 6.85. + -. 0.12mg/dl, CHO-OST311RQH group: 3.91. + -. 0.23mg/dl (p < 0.001, relative to CHO-ras clone-1 group), CHO-OST311RQ Δ 9 group: 4.33. + -. 0.15(p < 0.001, relative to CHO-ras clone-1 group)), which was comparable to the cell transplant group expressing full-length recombinants.

These results show that even though SEQ ID NO: 2 from at least amino acid 25Tyr to 33 Gly, and the biological activity of OST311 is intact.

EXAMPLE 27 examination of E.coli producing recombinant OST311

(1) Construction of OST311 E.coli expression vector OST311/pET3a

The following primers were synthesized.

OST311N：TGTATCCCAATGCCTCCCCACTG(SEQ ID NO：60)

OST311Bm：ATGGATCCCTAGATGAACTTGGCGAAGGG(SEQ ID NO：61)

PCR was performed using the OST311/pCAGGS plasmid prepared in example 19 as a template, OST311N (SEQ ID NO: 60) and OST311Bm (SEQ ID NO: 61) primers and pfu DNA polymerase (Promega, USA). After maintaining the temperature at 94 ℃ for 1 minute, 35 cycles of reaction were carried out, each cycle consisting of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute. After completion of the reaction, phenol/chloroform treatment was performed to inactivate the enzyme, and then DNA was recovered by ethanol precipitation. The DNA was digested with BamHI, and the objective OST311cDNA fragment was isolated by 2% agarose gel electrophoresis and then recovered with GeneClean II (BIO101, USA). At the same time, the plasmid vector pET3a (Novagen, USA) was digested with NdeI and then blunt-ended with Klenow fragment (Roche, Swiss). The vector was further digested with BamHI, and the resulting plasmid DNA fragment was separated by 0.8% agarose gel electrophoresis and then recovered with Gene Clean II (BIO101, USA). The resulting OST311cDNA fragment was ligated to the digested plasmid pET3a using the DNA ligation kit, version 2(TAKARA SHUZO, Japan). Then, the product was introduced into E.coli DH 5. alpha. for cloning, and plasmids were extracted. The nucleotide sequence of the plasmid was determined to ensure that the OST311cDNA had been inserted into pET3a as expected. This plasmid was designated OST311/pET3 a.

(2) Construction of OST311/pET28 vector for expression of OST311 in E.coli

The following primers were synthesized.

OST311Nd：ATCATATGTATCCCAATGCCTCCCCACTG(SEQ ID NO：62)

OST311Not：ATGCGGCCGCCTAGATGAACTTGGCGAAGGG(SEQ ID NO：63)

PCR was performed using OST311/pET3a plasmid as a template, OST311Nd (SEQ ID NO: 62) and OST311Not (SEQ ID NO: 63) primers and LA Taq (TAKARA SHUZO, Japan). After maintaining the temperature at 94 ℃ for 1 minute, 35 cycles of reaction were carried out, each cycle consisting of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute. After completion of the reaction, phenol/chloroform treatment was performed to inactivate the enzyme, and then the amplified DNA fragment was recovered by ethanol precipitation. The DNA was digested with NdeI and NotI, and the target OST311cDNA fragment was separated by 2% agarose gel electrophoresis and recovered by Gene Clean II (BIO101, USA). Meanwhile, the plasmid vector pET28(Novagen, USA) was digested with NdeI and NotI, and then dephosphorylated with bovine small intestine alkaline phosphatase (TAKARASHUZO, Japan). The products were then separated by 0.8% agarose gel electrophoresis, and the digested plasmid was recovered using Gene Clean II (BIO101, USA). The resulting OST311cDNA was ligated to the digested plasmid pET28 using the DNA ligation kit, version 2(TAKARA SHUZO, Japan). The ligation product was then introduced into E.coli DH 5. alpha. for cloning, and the plasmid was extracted. Determination of the nucleotide sequence of the plasmid ensured that the OST311cDNA, which was capable of expressing recombinant OST311 with His tag sequence added to the N-terminus, had been inserted into pET 28. This plasmid was designated OST311/pET 28. The amino acid sequence and the nucleotide sequence of the recombinant His-OST311 coded by the vector are shown in figure 26.

(3) Expression and production of recombinant His-OST311 in E.coli

The plasmid OST311/pET28 was introduced into E.coli BL21(DE3) Codon PlusRP (STRATAGENE, USA) for transformation, and then a clone was obtained. The obtained E.coli clone was inoculated into 100ml of LB medium containing 10mg of kanamycin (SIGMA, USA), and cultured overnight at 37 ℃. The bacterial cell suspension was inoculated into 1L of LB medium to A600 ═ 0.1, and then cultured with shaking in a 3L Sakaguchi flask at 37 ℃. The light absorption of the culture broth was measured over time. When a600 ═ 0.6 to 1.0 was achieved, isopropyl-1-thio- β -galactoside (IPTG) (Wako Pure Chemical Industries, Japan) was added to 1 mM. After 4 hours, the cells were recovered by centrifugation (7700gx15 min). The recovered cells were suspended in 20ml of 0.1M Tris-hydrochloric acid buffer (pH7.5) containing 1mM DTT, and then disrupted by French Press. The solution containing the disrupted cells was centrifuged (7700gx15 min), and the pellet was suspended in 15ml of 0.1M Tris-hydrochloric acid buffer (pH 7.5). DNaseI (Roche, Swiss) was added to the suspension to 0.1mg/ml, followed by shaking at 4 ℃ for 1 hour. Next, centrifugation (23400gx15 minutes) was performed, and then the precipitated fraction was collected as inclusion bodies. The resulting inclusion bodies were suspended in 10ml of 20mM Tris hydrochloric acid buffer (pH8) containing 0.75M urea and 1% Triton-X to be washed, and then centrifuged (23400gX15 minutes) to collect the precipitates. The washing process was repeated twice.

The washed inclusion bodies were suspended in 5ml of a denaturing solution (50mM phosphate buffer solution (pH8) containing 1mM DTT and 6M guanidine hydrochloride), and then dissolved at 37 ℃ for 1 hour with shaking. Insoluble material forming the precipitate was removed by centrifugation (23400gx15 min) and the solution was then equilibrated with 50mM phosphate buffer (pH6) containing 6M guanidine hydrochloride. The solubilized sample was applied to a column packed with Ni-NTA Agrose (QIAGEN, Germany), and then washed with 50mM phosphate buffer (pH6) containing 6M guanidine hydrochloride. The protein adsorbed to the column was eluted with 50mM phosphate buffer (pH4.5) containing 500mM imidazole (NacalaiTesque, Japan) and 6M guanidine hydrochloride, thereby purifying denatured His-OST 311. The concentration of the purified sample was known from its UV absorption at 280nm, and then a solution to denatured His-OST311 was prepared by adding 50mM phosphate buffer solution (pH6) containing 6M guanidine hydrochloride to the sample to a final concentration of 2 mg/ml. Cysteine was added as a reducing agent to the sample to a final concentration of 1mM and diluted 100-fold with 20mM phosphate buffer (pH6) containing 0.6M guanidine hydrochloride and 0.1% Tween20 for refolding. The incubation is carried out at 4 ℃ for 3 days or more.

The refolding solution was dialyzed against 0.1M acetate buffer (pH4.8) at 4 ℃. The dialyzed refolded solution was concentrated about 10-fold using ultrafiltration membrane and then purified by HPLC using cation exchange column SP-5PW (TOSOH, Japan). The protein was eluted with a linear NaCl gradient from 0.5M to 2M using 20mM phosphate buffer (pH6) containing glycerol. The elution pattern is shown in FIG. 27. SDS-PAGE analysis and mass spectrometry showed that His-OST311 was contained in the peaks eluted at lower salt concentrations in the two protein species eluted. As described above, about 0.6mg of the final purified product, His-OST311, can be prepared from about 1L of cultured cells.

(4) Construction of pET22b-MK-OST311 vector expressing MK-OST311

The following primers were synthesized.

OST311MK1：gaattcatatgaaatacccgaacgcttccccgctgctgggctccagctg(SEQ ID NO：64)

OST311MK2：cccaagcttgcggccgcctagatgaacttggc(SEQ ID NO：65)

Using the above His-OST311 expression plasmid OST311/pET28 as a template, OST311MK1(SEQ ID NO: 64) and OST311MK2(SEQ ID NO: 65) as primers, a target sequence was amplified by PCR. In the OST311cDNA obtained by this process, 27 nucleotides after the initiation codon (ATG) have been converted into the codon of E.coli. The PCR product was purified using QIAquick PCR purification kit (QIAGEN, Germany), and then digested with restriction enzymes NdeI (TAKARA SHUZO, Japan) and NotI (TAKARA SHUZO, Japan) at 37 ℃ for 1 hour. The digested PCR products were separated by agarose electrophoresis and then purified using QIAquick PCR purification kit (QIAGEN, Germany). The resulting DNA fragment was digested with restriction enzymes NdeI and NotI at 37 ℃ for 1 hour, and then ligated with a plasmid vector pET22b (Novagen, USA) separated and purified by agarose electrophoresis at 16 ℃ for 15 minutes using a DNA ligation kit Ver2(TAKARASHUZO, Japan). The ligated product was introduced into Escherichia coli JM109(TAKARA SHUZO, Japan) for cloning, and then the plasmid was extracted by a standard method. The nucleotide sequence of the resulting plasmid was determined to ensure that the resulting OST311cDNA had been inserted into the pET22b vector. This plasmid was designated pET22-MK-OST 311. The nucleotide sequence and amino acid sequence of recombinant MK-OST311 encoded by the vector are shown in FIG. 26.

(5) Expression and production of MK-OST311 in E.coli

The plasmid pET22-MK-OST311 was introduced into E.coli BL21(DE3) Codon PlusRP (STRATAGENE, USA), and then a transformed clone was obtained. The resulting E.coli clone was inoculated into 100ml of LB medium containing 10mg of ampicillin, and then cultured overnight at 37 ℃. The cell suspension of the bacteria was inoculated in 1L of LB medium to A600 ═ 0.1, and then shake-cultured at 37 ℃ using a 3L Sakaguchi flask. IPTG was added to the bacterial cell culture solution to induce expression of the recombinant, and then inclusion bodies were prepared in a manner similar to the above method for preparing His-OST 311.

The washed inclusion bodies were suspended in 5ml of a denaturing solution (50mM phosphate buffer solution (pH8) containing 1mM DTT and 6M guanidine hydrochloride), and the suspension was then dissolved by shaking at 37 ℃ for 1 hour. The solubilized product was diluted 2-fold with denaturing solution and then 100-fold with 20mM phosphate buffer containing 0.6M guanidine hydrochloride and 0.1% Tween20 to begin refolding. Incubate at 4 ℃ for 3 days or more. It is known that proteins precipitate under conditions of addition of an oxidizing agent and pH of 7 or more and thus the efficiency of refolding decreases significantly. The refolding solution was dialyzed against 0.1M acetate buffer (pH4.8) at 4 ℃. The dialyzed refolded solution was concentrated about 10-fold using ultrafiltration membrane and then purified by HPLC using cation exchange column SP-5PW (TOSOH, Japan). The protein was eluted with a linear NaCl gradient from 0.5M to 2M using 20mM phosphate buffer (pH6) containing 10% glycerol. As shown in FIG. 28, there are two protein elution peaks, and SDS-PAGE analysis and mass spectrometry analysis show that MK-OST311 is contained in the peak eluted at the lower salt concentration. Thus, about 0.6mg of the final purified product, MK-OST311, can be prepared from about 1L of cultured cells.

(6) PEGylation of MK-OST311

10ml of ion-exchange column-purified MK-OST311(0.05mg/ml) was adjusted to pH4.8 with 10% acetic acid. To this solution, 25mg of activated PEG (Sharewater, USA) with a molecular weight of 20000 dissolved in 10mM acetate buffer (pH4.8) was added with stirring. After 15 minutes, 1M sodium cyanoborohydride (Nacalai Tesque, Japan) dissolved in 10mM acetic acid buffer (pH4.8) was added to the solution to a final concentration of 15mM, and the solution was stirred at 4 ℃ for 16 hours. OST311 PEGylated by this reaction was purified by HPLC using cation exchange column SP-5PW (TOSOH, Japan). The protein was eluted with a linear NaCl gradient from 0.5M to 2M using 20mM phosphate buffer (pH6) containing 10% glycerol. As shown in FIG. 29, PEGylated MK-OST311 elutes as a peak at lower ionic strength compared to MK-OST 311.

(7) Detection of recombinant Activity of His-OST311

To examine the biological activity of the purified His-OST311 recombinant, the recombinant protein was administered once to normal mice (5 weeks old, BALB/c, male, six per group) at a dose of 4.5ug/0.1ml via the tail vein in the manner described in example 24. After 9 hours, serum phosphate and 1, 25-dihydroxyvitamin D3 levels were measured by a similar method as described in example 20. A positive control group was subjected to a single administration of the same dose of the purified recombinant obtained from CHO-OST311H cells, and a vehicle-administered group was also subjected to a single administration of a vehicle comprising 20mM phosphate buffer (pH6.9) and 0.3M NaCl in an amount of 0.1ml each, both groups being administered via the tail vein.

As shown in fig. 30A, a significant effect of reducing serum phosphate levels was observed in the group administered His-OST311, compared to the group administered with the vehicle, 9 hours after the administration. The degree of reduction was comparable to the CHO-generated recombinant protein-administered group. In addition, as shown in fig. 32, the serum 1, 25-dihydroxyvitamin D3 level in the His-OST 311-administered group was also significantly decreased after 9 hours of administration.

As described in examples 24(3) and (4), a significant decrease in serum 1, 25-dihydroxyvitamin D3 levels was observed 4 hours after one administration of OST311 protein produced by CHO cells. Before this time, 1 hour after the administration, a decrease in the expression of 25-hydroxyvitamin D-1-alpha-hydroxylase (1. alpha. OHAse) and an increase in the expression of 25-hydroxyvitamin D-24-hydroxylase (24OHAse) were observed in the kidney. Therefore, His-OST311 was administered to BALB/c mice (5 weeks old, male) once per 4.5ug/0.1ml via the tail vein, and then the kidneys were excised after 1 and 4 hours. Changes in the expression of 1 α OHase and 24OHase in the kidney were analyzed by Northern blotting. A positive control group was subjected to a single administration of the same dose of the purified recombinant obtained from CHO-OST311H cells, and a vehicle-administered group was also subjected to a single administration of a vehicle comprising 20mM phosphate buffer (pH6.9) and 0.3M NaCl in an amount of 0.1ml each, both groups being administered via the tail vein. As shown in FIG. 31, His-OST311 caused a decrease in the expression of 1. alpha. OHAse gene and an increase in the expression of 24OHAse gene1 hour after administration, similar to the CHO-produced recombinants. This revealed that His-OST311 has activity comparable to that of CHO-produced recombinants, and can regulate the expression of vitamin D metabolizing enzyme genes. Furthermore, as shown in fig. 32, 4 hours after administration of the recombinant, serum 1, 25-dihydroxyvitamin D3 levels showed a moderate decrease, with a more marked decrease at 8 hours. This revealed that the manner of this change was almost identical to that observed in the experiment described in example 24(3) for recombinant production given CHO.

From the above results, it was revealed that recombinant His-OST311 produced by E.coli has at least serum phosphate-lowering activity and vitamin D metabolism-regulating activity, which is comparable to the activity of a secretory recombinant produced by CHO-OST311H cells.

(8) Detection of Activity of PEGylated MK-OST311

The biological activity of PEGylated MK-OST311 was measured by administering PEGylated MK-OST311 once per 5.0ug/0.1ml to normal mice (5 weeks old, BALB/c, males, 8 per group) via the tail vein in the manner described in example 24, and then measuring serum phosphate levels 9 hours after administration by a method similar to example 20. A single administration of a vector comprising 20mM PB (pH6.0), 10% glycerol, 1M NaCl and 0.1% Tween was also given to one vector administration group via the tail vein in a single dose in an amount of 0.1 ml/mouse. At 8 hours after the administration, blood was collected from the eye socket using a glass capillary, and then the level of inorganic phosphate in the resulting serum was measured. As shown in FIG. 30B, a significant effect of reducing serum phosphate levels was observed in the group administered PEG MK-OST 311. This result revealed that the recombinant produced by E.coli was not inhibited in its biological activity when it was PEGylated.

Example 28 introduction of amino acid mutations into cleavage sites

As described in example 9, OST311 is shown in SEQ ID NO: 2 between amino acid residues 179(Arg) and 180 (Ser). Meanwhile, in example 19, it was confirmed that simultaneous substitution of the amino acid residues at positions 176(Arg) and 179(Ser) of OST311 with Gln inhibited the cleavage. These facts suggest the possibility that the cleavage is due to certain proteases recognizing motifs containing adjacent RXXR and RRXXR sequences. Moreover, the in vivo administration of a peptide consisting of SEQ ID NO: 4, the same or similar cleavage as described above may occur. Thus, SEQ ID NO: 2 with Ala, Gln and Trp, and then examined how these substitutions affect the expression and secretion patterns of the mutant recombinants in CHO cells.

(1) Construction of OST311 Gene having a mutation in cleavage site

The following primers were synthesized.

pyh23PA1F AACACCCCCATAGCACGGCGGCACA(SEQ ID NO：66)

pyh23PA1R TGTGCCGCCGTGCTATGGGGGTGTT(SEQ ID NO：67)

pyh23RA1F ACCCCCATACCAGCGCGGCACACCCG(SEQ ID NO：68)

pyh23RA1R CGGGTGTGCCGCGCTGGTATGGGGGT(SEQ ID NO：69)

pyh23RA2F CCCATACCACGGGCGCACACCCGGAG(SEQ ID NO：70)

pyh23RA2R CTCCGGGTGTGCGCCCGTGGTATGGG(SEQ ID NO：71)

pyh23HA1F ATACCACGGCGGGCCACCCGGAGCGC(SEQ ID NO：72)

pyh23HA1R GCGCTCCGGGTGGCCCGCCGTGGTAT(SEQ ID NO：73)

pyh23TA1F CCACGGCGGCACGCCCGGAGCGCCG(SEQ ID NO：74)

pyh23TA1R CGGCGCTCCGGGCGTGCCGCCGTGG(SEQ ID NO：75)

pyh23RA3F CGGCGGCACACCGCGAGCGCCGAGGA(SEQ ID NO：76)

pyh23RA3R TCCTCGGCGCTCGCGGTGTGCCGCCG(SEQ ID NO：77)

pyh23SA1F CGGCACACCCGGGCCGCCGAGGACGA(SEQ ID NO：78)

pyh23SA1R TCGTCCTCGGCGGCCCGGGTGTGCCG(SEQ ID NO：79)

pyh23RKQ1F ACCCCCATACCACAGCGGCACACCCG(SEQ ID NO：80)

pyh23RKQ1R CGGGTGTGCCGCTGTGGTTGGGGGT(SEQ ID NO：81)

pyh23RKQ2F CCCATACCACGGCAGCACACCCGGAG(SEQ ID NO：82)

pyh23RKQ2R CTCCGGGTGTGCTGCCGTGGTATGGG(SEQ ID NO：83)

pyh23RKQ3F CGGCGGCACACCCAGAGCGCCGAGGA(SEQ ID NO：84)

pyh23RKQ3R TCCTCGGCGCTCTGGGTGTGCCGCCG(SEQ ID NO：85)

pyh23RWF CGGCGGCACACCTGGAGCGCCGAGG(SEQ ID NO：86)

pyh23RWR CCTCGGCGCTCCAGGTGTGCCGCCG(SEQ ID NO：87)

pyh23PA1F and pyh23PA1R are forward and reverse primers for introducing mutations in which the substitution of guanine for the 652 th cytosine of the OST311cDNA (SEQ ID NO: 1) results in the amino acid sequence of SEQ ID NO: 2 (Pro) is replaced by Ala. Hereinafter, this mutation is referred to as P174A.

pyh23RA1F and pyh23RA1R are forward and reverse primers for introducing mutations in which replacement of 655 cytosine and 656 guanine of OST311cDNA (SEQ ID NO: 1) with guanine and cytosine, respectively, results in SEQ ID NO: 2 by Ala. Hereinafter, this mutation is referred to as R175A.

pyh23RA2F and pyh23RA2R are forward and reverse primers for introducing mutations in which 658 cytosine and 659 guanine of OST311cDNA (SEQ ID NO: 1) is replaced with guanine and cytosine, respectively, resulting in SEQ ID NO: 2 by Ala. Hereinafter, this mutation is referred to as R176A.

pyh23HA1F and pyh23HA1R are forward and reverse primers for introducing mutations in which 661 cytosine and 662 adenine of OST311cDNA (SEQ ID NO: 1) are replaced with guanine and cytosine, respectively, resulting in SEQ ID NO: 2 (His) was substituted with Ala. Hereinafter, this mutation is referred to as H177A.

pyh23TA1F and pyh23TA1R are forward and reverse primers for introducing mutations in which replacement of 664 adenine of OST311cDNA (SEQ ID NO: 1) with guanine results in SEQ ID NO: 2 (Thr) is substituted with Ala. Hereinafter, this mutation is referred to as T178A.

pyh23RA3F and pyh23RA3R are forward and reverse primers for introducing mutations in which the replacement of 667 cytosine and 668 guanine of OST311cDNA (SEQ ID NO: 1) with guanine and cytosine, respectively, results in the amino acid sequence of SEQ ID NO: 2 by Ala. Hereinafter, this mutation is referred to as R179A.

pyh23SA1F and pyh23SA1R are forward and reverse primers for introducing mutations in which 670-adenine and 671-guanine of OST311cDNA (SEQ ID NO: 1) are replaced with guanine and cytosine, respectively, resulting in SEQ ID NO: 2 (Ser) is replaced by Ala. Hereinafter, this mutation is referred to as S180A.

pyh23RKQ1F and pyh23RKQ1R are forward and reverse primers for introducing mutations in which replacement of 656 guanine of the OST311cDNA (SEQ ID NO: 1) with adenine results in SEQ ID NO: 2 by Gln. Hereinafter, this mutation is referred to as R175Q.

pyh23RKQ2F and pyh23RKQ2R are forward and reverse primers for introducing mutations in which the substitution of 659 guanine of the OST311cDNA (SEQ ID NO: 1) with adenine results in the amino acid sequence of SEQ ID NO: 2 by Gln. Hereinafter, this mutation is referred to as R176Q.

pyh23RKQ3F and pyh23RKQ3R are forward and reverse primers for introducing mutations in which the replacement of 668 guanine of the OST311cDNA (SEQ ID NO: 1) with adenine results in SEQ ID NO: 2 by Gln. Hereinafter, this mutation is referred to as R179Q.

pyh23RWF and pyh23RWR are forward and reverse primers for introducing mutations in which the substitution of 659 cytosine of the OST311cDNA (SEQ ID NO: 1) with thymine results in the amino acid sequence of SEQ ID NO: 2 (Arg) is substituted with Trp. Hereinafter, this mutation is referred to as R179W.

(1) Construction of-1 OST311P174AH Gene

Two reaction solutions (100 ul each) were prepared using Pyrobest DNA polymerase (TAKARA SHUZO, Japan) according to the instructions of the attached manufacturer. One reaction solution, OST311ME1(SEQ ID NO: 45) and pyh23PA1F (SEQ ID NO: 66) were used as primers at a final concentration of 0.2uM, and for the other reaction solution, pyh23PA1R (SEQ ID NO: 67) and OST311HNt (SEQ ID NO: 46) were used as primers at a final concentration of 0.2 uM. To each reaction solution, 10ng of the OST311/pCAGGS plasmid described in example 19(1) was added as a template, and then the reaction solution was maintained at 94 ℃ for 1 minute. Then 40 PCR reactions were performed, each cycle consisting of 94 ℃ for 20 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute. These two reaction solutions were diluted 10-fold, respectively, and 1ul of them was prepared and added to 100ul of the reaction solution prepared according to the document attached to Pyrobest DNA polymerase (TAKARA SHUZO, Japan). OST311ME1(SEQ ID NO: 45) and OST311HNt (SEQ ID NO: 46) were added to a final concentration of 0.2uM as primers, and the solution was then maintained at 94 ℃ for 1 minute. Then, 30 PCR reactions were performed, each cycle consisting of 94 ℃ for 20 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute and 30 seconds. After the PCR reaction, the solution was kept at 72 ℃ for 7 minutes. The reaction product thus obtained was collected using Gene Clean II (BIO101, USA) according to the attached manufacturer's instructions. The product was then digested with EcoRI and NotI, followed by 2% agarose gel electrophoresis to isolate a DNA fragment of approximately 800 bp. The fragment was collected using Gene CleanII (BIO101, USA). The resulting DNA fragment was inserted into EcoRI and NotI sites of pEAK8 vector (EdgeBio, USA), thereby obtaining plasmid OST311P174AH-pEAK 8. Plasmid DNA was prepared according to a standard method and its nucleotide sequence was determined using ABI3700 fluorescent DNA sequencer (PE Applied Systems, USA). This confirmed that the mutation P174H was introduced. Furthermore, it was confirmed that a histidine tag was added to the C-terminus. The polypeptide encoded by the mutant gene containing P174A introduced herein is referred to as OST311P174 AH.

(1) Preparation of-2 OST311R175AH Gene

The OST311R175AH gene was prepared by a method similar to (1) -1 using pyh23RA1F (SEQ ID NO: 68) and pyh23RA1R (SEQ ID NO: 69) as primers.

(1) Preparation of-3 OST311R176AH Gene

The OST311R176AH gene was prepared by a method similar to (1) -1 using pyh23RA2F (SEQ ID NO: 70) and pyh23RA2R (SEQ ID NO: 71) as primers.

(1) Preparation of-4 OST311H177AH Gene

The OST311H177AH gene was prepared by a method similar to (1) -1 using pyh23HA1F (SEQ ID NO: 72) and pyh23HA1R (SEQ ID NO: 73) as primers.

(1) Preparation of-5 OST311T178AH Gene

The OST311T178AH gene was prepared by a method similar to (1) -1 using pyh23TA1F (SEQ ID NO: 74) and pyh23TA1R (SEQ ID NO: 75) as primers.

(1) Preparation of-6 OST311R179AH Gene

The OST311R179AH gene was prepared by a method similar to (1) -1 using pyh23RA3F (SEQ ID NO: 76) and pyh23RA3R (SEQ ID NO: 77) as primers.

(1) Preparation of-7 OST311S180AH Gene

The OST311S180AH gene was prepared by a method similar to (1) -1 using pyh23SA1F (SEQ ID NO: 78) and pyh23SA1R (SEQ ID NO: 79) as primers.

(1) Preparation of-8 OST311R175QH Gene

The OST311R175QH gene was prepared by a method similar to (1) -1 using pyh23RKQ1F (SEQ ID NO: 80) and pyh23RKQ1R (SEQ ID NO: 81) as primers.

(1) Preparation of-9 OST311R176QH Gene

The OST311R176QH gene was prepared by a method similar to (1) -1 using pyh23RKQ2F (SEQ ID NO: 82) and pyh23RKQ2R (SEQ ID NO: 83) as primers.

(1) Preparation of-10 OST311R179QH Gene

The OST311R179QH gene was prepared by a method similar to (1) -1 using pyh23RKQ3F (SEQ ID NO: 84) and pyh23RKQ3R (SEQ ID NO: 85) as primers.

(1) Preparation of-11 OST311R179WH Gene

The OST311R179WH gene was prepared by a method similar to (1) -1 using pyh23RWF (SEQ ID NO: 86) and pyh23RWR (SEQ ID NO: 87) as primers.

(2) Transient expression of the cleavage site-mutated OST311 Gene and preparation of the regulatory Medium

pEAK rapid cells (Edgebiosystems, USA) were seeded into 12-well plates. Cells were transfected with the above 11 expression plasmids for mutant OST311 by the calcium phosphate method according to the file attached to the pEAK system (EdgeBiosystems, USA). The cells were left for 4 hours, the medium was replaced with 1.5ml of serum-free MEM α medium, the cells were cultured at 37 ℃ for 2 days, and then the conditioned medium was collected.

(3) Evaluation of expression of OST311 Gene having a cleavage site mutation

The resulting conditioned medium was subjected to Western blotting in a similar manner as described in example 6(3), and then the conditioned medium was examined for the presence of recombinant OST311 having a mutation in the cleavage site. OST 311-specific polyclonal antibody, 311-148 described in example 22 was used for the detection. Thus, as shown in FIG. 33, similar to the wild type, OST311 recombinants introducing mutations P174A, R175A, R175Q, H177A, T178A or S180A among the substitutions occurring at amino acid residues 174 to 180 observed a peptide containing the amino acid sequence shown in SEQ ID NO: 6. However, no degradation products were observed in OST311 recombinants introducing mutations R176A, R179A, R176Q, R179A or R179W. These results suggest that, in particular, amino acid residues 176(Arg) and 179(Arg) play an important role for the cleavage that occurs between amino acid residues 179(Arg) and 180 (Ser). That is, at least when these two residues are substituted with any of Ala, Gln or Trp, cleavage can be eliminated or inhibited. Thus, the use of this finding can facilitate the production of full-length polypeptides.

Example 29

As shown in example 6, the recombinant product obtained by expressing OST311 in CHO cells or COS cells was cleaved between 179(Arg) and 180(Ser) amino acid residues located after the RXR motif shown in example 9. Example 18 clearly shows that the hypophosphatemia-inducing activity of OST311 protein is retained in the full-length protein that is not cleaved at this site. In addition, it is clear from the mutagenesis experiments shown in examples 19 and 28 that RXXR motifs are involved in this cleavage. To efficiently produce and obtain recombinant OST311, it is an important issue to avoid this cleavage, and introduction of mutations in RXXR motifs as shown in example 28 is one of the effective methods. Furin is a protease known to recognize RXXR motifs. The enzyme is located outside the golgi apparatus and is thought to cleave post-translational proteins by recognizing RXXR motifs during secretion.

(1) Furin is involved in the cleavage of OST311

The OST311/pCAGGS plasmid was introduced into furin-deficient LoVo cells using Transfectum (Promega, USA). The cells were then cultured for 8 days. When OST311 protein transiently expressed and secreted into the culture broth was analyzed by Western blotting using the 311-148 antibody, no cleaved product was detected. This result indicates that furin is involved in the observed cleavage of OST311 when OST311 is produced.

(2) Avoiding cutting of OST311

From the above results, it is concluded that inhibition of the furin activity is effective in promoting the production of OST311 without cleavage between amino acids 179(Arg) and 180 (Ser). Accordingly, it is conceivable to express OST311 in a host such as LoVo cells which does not have a furin enzyme activity, or under conditions in which the furin enzyme activity is inhibited by adding a furin alcohol inhibitor. To inhibit the furin activity of CHO-OST311H cells, α 1-antitrypsin Portland (α 1-PDX) was transiently expressed according to the method reported by Benjannet S et al (J Biol Chem 272: 26210-8, 1997), and the conditioned medium was collected. The ratio of full-length polypeptide in the recombinant product was increased compared to the conditioned medium of control CHO-OST311H cells without the gene. Accordingly, it can be concluded that the production efficiency of full-length OST311 protein can be improved by introducing an agent inhibiting the activity of furin externally or internally.

All publications, patents and patent applications cited herein are incorporated by reference in their entirety.

Industrial applications

According to the present invention, there are provided a polypeptide that regulates phosphate metabolism, calcium metabolism and/or calcification, a DNA encoding the polypeptide, a pharmaceutical composition containing the polypeptide as an active ingredient, an antibody recognizing the polypeptide, a pharmaceutical composition containing the antibody as an active ingredient, a diagnostic method and a diagnostic composition using the antibody.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 12: synthesis of DNA

SEQ ID NO: 13: synthesis of DNA

SEQ ID NO: 14: synthesis of DNA

SEQ ID NO: 15: synthesis of DNA

SEQ ID NO: 16: synthesis of DNA

SEQ ID NO: 17: synthesis of DNA

SEQ ID NO: 18: synthesis of DNA

SEQ ID NO: 19: synthesis of DNA

SEQ ID NO: 20: synthesis of DNA

SEQ ID NO: 21: synthesis of DNA

SEQ ID NO: 22: synthesis of DNA

SEQ ID NO: 23: synthesis of DNA

SEQ ID NO: 24: synthesis of DNA

SEQ ID NO: 25: synthesis of DNA

SEQ ID NO: 26: synthesis of DNA

SEQ ID NO: 27: synthesis of DNA

SEQ ID NO: 28: synthetic peptides

SEQ ID NO: 29: synthetic peptides

SEQ ID NO: 30: synthesis of DNA

SEQ ID NO: 31: synthesis of DNA

SEQ ID NO: 32: synthesis of DNA

SEQ ID NO: 33: synthesis of DNA

SEQ ID NO: 34: synthesis of DNA

SEQ ID NO: 35: synthesis of DNA

SEQ ID NO: 36: synthesis of DNA

SEQ ID NO: 37: synthesis of DNA

SEQ ID NO: 38: synthesis of DNA

SEQ ID NO: 39: synthesis of DNA

SEQ ID NO: 40: synthesis of DNA

SEQ ID NO: 41: synthesis of DNA

SEQ ID NO: 42: synthesis of DNA

SEQ ID NO: 43: synthesis of DNA

SEQ ID NO: 44: synthesis of DNA

SEQ ID NO: 45: synthesis of DNA

SEQ ID NO: 46: synthesis of DNA

SEQ ID NO: 47: synthesis of DNA

SEQ ID NO: 48: synthesis of DNA

SEQ ID NO: 49: synthetic peptides

SEQ ID NO: 50: synthetic peptides

SEQ ID NO: 51: synthetic peptides

SEQ ID NO: 52: synthetic peptides

SEQ ID NO: 53: synthesis of DNA

SEQ ID NO: 54: synthesis of DNA

SEQ ID NO: 55: synthesis of DNA

SEQ ID NO: 56: synthesis of DNA

SEQ ID NO: 57: synthesis of DNA

SEQ ID NO: 58: synthesis of DNA

SEQ ID NO: 59: synthesis of DNA

SEQ ID NO: 60: synthesis of DNA

SEQ ID NO: 61: synthesis of DNA

SEQ ID NO: 62: synthesis of DNA

SEQ ID NO: 63: synthesis of DNA

SEQ ID NO: 64: synthesis of DNA

SEQ ID NO: 65: synthesis of DNA

SEQ ID NO: 66: synthesis of DNA

SEQ ID NO: 67: synthesis of DNA

SEQ ID NO: 68: synthesis of DNA

SEQ ID NO: 69: synthesis of DNA

SEQ ID NO: 70: synthesis of DNA

SEQ ID NO: 71: synthesis of DNA

SEQ ID NO: 72: synthesis of DNA

SEQ ID NO: 73: synthesis of DNA

SEQ ID NO: 74: synthesis of DNA

SEQ ID NO: 75: synthesis of DNA

SEQ ID NO: 76: synthesis of DNA

SEQ ID NO: 77: synthesis of DNA

SEQ ID NO: 78: synthesis of DNA

SEQ ID NO: 79: synthesis of DNA

SEQ ID NO: 80: synthesis of DNA

SEQ ID NO: 81: synthesis of DNA

SEQ ID NO: 82: synthesis of DNA

SEQ ID NO: 83: synthesis of DNA

SEQ ID NO: 84: synthesis of DNA

SEQ ID NO: 85: synthesis of DNA

SEQ ID NO: 86: synthesis of DNA

SEQ ID NO: 87: synthesis of DNA

Sequence listing

Claims

1. A DNA encoding the following polypeptide (a), (b), (c) or (d):

(c) consisting of SEQ ID NO: 2, wherein the partial sequence contains at least the amino acids 34 to 201 of the amino acid sequence, or

(d) Consisting of SEQ ID NO: 2, wherein the partial sequence:

(i) an amino acid sequence containing the 34 th to 201 th amino acids in the amino acid sequence,

2. A DNA comprising the following DNA (e) or (f):

(e) consisting of SEQ ID NO: 1 from nucleotide 133 to 885, or a DNA consisting of the nucleotide sequence of SEQ ID NO: 3 from nucleotide 1 to 681 in the nucleotide sequence shown in (a), or

3. A recombinant vector comprising the DNA of claim 1or 2.

4. A transformant containing the recombinant vector of claim 3.

5. A pharmaceutical composition comprising the following polypeptide (a), (b), (c), (D) or (m) as an active ingredient and being effective against at least one symptom selected from hyperphosphatemia, hyperparathyroidism, renal osteodystrophy, ectopic calcification, osteoporosis and vitamin D hyperemia,

(a) consisting of SEQ ID NO: 2 or 4;

(d) Consisting of SEQ ID NO: 2, wherein the partial sequence:

(i) consisting of an amino acid sequence from the 34 th to the 201 st amino acid in the above amino acid sequence,

(iii) has hypophosphatemia inducing activity, phosphate transport inhibiting activity, calcification inhibiting activity or in vivo vitamin D metabolism regulating activity

(m) the polypeptide of the above (a) to (d) modified with at least one selected from the group consisting of polyethylene glycol, dextran, poly (N-vinyl-pyrrolidone), polypropylene glycol homopolymer, copolymer of polypropylene oxide and polyethylene oxide, polyoxyethylated polyol and polyvinyl alcohol.

6. A method for producing an antibody, which comprises the step of immunizing an animal with the polypeptide (a), (b), (c), (d) or (m), or a partial fragment thereof as an antigen

(a) Consisting of SEQ ID NO: 2 or 4;

(d) Consisting of SEQ ID NO: 2, wherein the partial sequence:

7. A pharmaceutical composition comprising, as an active ingredient, an antibody reactive with the following polypeptide (a), (b), (c), (d) or (m), or a partial fragment thereof,

(a) consisting of SEQ ID NO: 2 or 4;

(d) Consisting of SEQ ID NO: 2, wherein the partial sequence:

8. A pharmaceutical composition comprising, as an active ingredient, an antibody reactive with the following polypeptide (a), (b), (c), (D) or (m), or a partial fragment thereof, and capable of regulating calcium metabolism, phosphate metabolism, calcification or vitamin D metabolism in vivo,

(a) consisting of SEQ ID NO: 2 or 4;

(d) Consisting of SEQ ID NO: 2, wherein the partial sequence:

9. A pharmaceutical composition comprising, as an active ingredient, an antibody reactive with the following polypeptide (a), (b), (c), (d) or (m), or a partial fragment thereof, and effective against bone diseases,

(a) consisting of SEQ ID NO: 2 or 4;

(d) Consisting of SEQ ID NO: 2, wherein the partial sequence:

10. The pharmaceutical composition of claim 9, wherein said bone disease is at least one of osteoporosis, vitamin D resistant rickets, renal osteodystrophy, dialysis related bone disease, calcium incompetent bone disease, paget's disease, and tumor-induced osteomalacia.

11. A diagnostic agent comprising, as an active ingredient, an antibody reactive with the following polypeptide (a), (b), (c), (D) or (m), or a partial fragment thereof, for use in a disease causing abnormality in at least one of abnormal calcium metabolism, abnormal phosphate metabolism, abnormal calcification and abnormal vitamin D metabolism,

(a) consisting of SEQ ID NO: 2 or 4;

(d) Consisting of SEQ ID NO: 2, wherein the partial sequence:

12. The diagnostic reagent of claim 11, wherein the bone disease is at least one disease of renal failure, renal phosphate leakage, renal tubular acidosis and fanconi syndrome salt.

13. A diagnostic reagent for bone diseases comprising an antibody reactive with the following polypeptide (a), (b), (c), (d) or (m), or a partial fragment thereof,

(a) consisting of SEQ ID NO: 2 or 4;

(d) Consisting of SEQ ID NO: 2, wherein the partial sequence:

14. The diagnostic reagent of claim 13, wherein said bone disease is a disease in which at least one of osteoporosis, vitamin D-resistant rickets, renal osteodystrophy, dialysis-related bone disease, calcified osteopathy, paget's disease, and tumor-induced osteomalacia is abnormal.

15. A diagnostic reagent comprising a peptide having SEQ ID NO: 11or a fragment thereof, and is useful for a disease that causes abnormality in at least one of abnormal calcium metabolism, abnormal phosphate metabolism, and abnormal calcification.

16. The diagnostic reagent of claim 15, wherein the partial fragment has the sequence of SEQ id no: 11 from nucleotide 498 to 12966.

17. The diagnostic reagent of claim 15 or 16, wherein the disease is autosomal dominant rickets/osteomalacia.