JP2009232732A - Variant fgf - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To discover gene mutation taking part in synostosis, to provide a method for judging onset risk of the disease based thereon, and to provide a variant FGF having modified affinity to sulfated glycosaminoglycan, and modified interstitial diffusibility. <P>SOLUTION: The method for judging the onset risk of the synostosis includes detecting the presence or absence of the substitution of Asn143 of FGF9 polypeptide with Thr, or the mutation of a codon encoding the Asn143 in Fgf9 gene to a codon encoding the Thr. The mutant FGF polypeptide in which the Asn taking part in hydrogen bond between monomers in an FGF homodimer is substituted with the Thr is also provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to mutant FGF and uses thereof. More specifically, the present invention relates to a risk of developing osteosynthesis characterized by detecting mutant FGF with reduced affinity for sulfated proteoglycan, a drug containing the mutant FGF, and the presence or absence of the mutant FGF. It is related with the determination method etc.

  Skeletal morphology generation is accompanied by coordinated generation of joints, and its position determines the layout of the skeleton. The joint is structurally and functionally heterogeneous. Synovial joints arise from the initial aggregation of chondrocytes and subsequently differentiate into separate skeletal structures separated by joint spaces. In contrast, the sutures were separated by a narrow area containing immature and rapidly dividing osteogenic precursors, some of which become osteoblasts and produce new bone Includes two bone plates.

  Gain-of-function mutations in the fibroblast growth factor (FGF) receptor result in chondrodysplasia and cranial suture early healing, highlighting the function of FGF signaling in both bone and joint development . In particular, radiohumeral and early cranial suture fusion in Antley-Bixler syndrome (ABS) involving a dominant mutation in fibroblast growth factor receptor 2 (Fgfr2) locus Co-occurring suggests the contribution of FGF signaling in both synovial joint and suture development (Non-Patent Document 1). A mouse with a similar syndrome-like joint defect expressing a gain-of-function form of Fgfr2c associated with Cluson syndrome (Non-patent Document 2) or a mouse expressing an ectopic and constitutively active FGFR1 kinase domain ( Non-patent document 3). These findings suggest that FGF signaling promotes cartilage formation and / or bone formation while inhibiting joint and suture development. Thus, spatiotemporal constraints on FGF signaling may achieve coordinated development of both bones and joints.

  Local FGF signaling is regulated at several different levels. First, there is a spatiotemporally restricted expression of FGF ligand. Among the 22 FGF ligands, FGF2, FGF4, FGF7, FGF8, FGF9, FGF10, FGF17 and FGF18 are expressed in the limb buds and the developing skeleton. Among these, the functional involvement of FGF2, FGF9, and FGF18 in cartilage formation and / or bone formation has been demonstrated by a loss-of-function mutation (Non-Patent Documents 4 to 7). Induction of chondrogenic phenotype by overexpression of FGF9 in mice has also demonstrated the ability of FGF9 to affect cartilage formation (Non-patent Document 8). Other elements involved in FGF signaling are heparan sulfate proteoglycans (HSPGs). Genetic studies in mice and Drosophila melanogaster suggest that HSPGs regulate FGF ligand distribution and receptor binding (Non-Patent Documents 9 and 10). In mice, hypomorphic mutations in Ext1, a key glycosyltransferase enzyme required for HSPG glycosaminoglycan chain synthesis, cause elbow and knee joint fusion (Non-Patent Document 11). In particular, the nerve-specific knockout of Ext1 revealed the need for heparan sulfate (HS) in the proper distribution of FGF8 during brain pattern formation. Furthermore, another HS biosynthetic gene, Ndst1, is required for FGF signaling during lens development. Together, these findings suggest that the FGF / HS interaction controls FGF signaling during organ development, including joint development. Finally, structural analysis of FGF9 suggests that FGF9 can form a homodimer that can affect signal transduction ability (Non-patent Documents 12 and 13). Since homodimerization of FGF9 closes several important receptor binding sites in FGF9, an autoinhibition mechanism may function to regulate FGF9-dependent signaling. However, the function of this proposed autoinhibition mechanism has not yet been demonstrated.

  As described above, it has been reported that various FGFs are involved in the development of joints and sutures, and that osteosynthesis is induced by a gain-of-function mutation of FGFR. It is not known at all that this mutation induces osteosynthesis. It is also not known that FGF mutations affect the affinity for sulfated glycosaminoglycans and their tissue diffusibility.

  The inventors have found that spontaneous mouse mutants, elbow-knee-synostosis (Eks), have ABS-like skeletal abnormalities, radiohumeral and tibiofemoral osteosynthesis, It has been previously reported that it exhibits early fusion of the skull suture and pulmonary hypoplasia (Non-patent Document 14).

On the other hand, Patent Document 1 discloses various FGF mutants. The document describes that FGF mutations induce changes in their receptor specificity. However, there is no description on the influence of FGF on tissue diffusivity and affinity with sulfated glycosaminoglycan.
WO02 / 036732 Reardon, W., Smith, A., Honour, JW, Hindmarsh, P., Das, D., Rumsby, G., Nelson, I., Malcolm, S., Ades, L., and Sillence, D. et al., (2000). Evidence for digenic inheritance in some cases of Antley-Bixler syndrome? J. Med. Genet. 37, 26-32. Eswarakumar, VP, Horowitz, MC, Locklin, R., Morriss-Kay, GM, and Lonai, P. (2004) .A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis.Proc. Natl Acad. Sci. USA. 101, 12555-12560. Wang, Q., Green, RP, Zhao, G., and Ornitz, DM (2001). Differential regulation of endochondral bone growth and joint development by FGFR1 and FGFR3 tyrosine kinase domains.Development 128, 3867-3876. Montero, A., Okada, Y., Tomita, M., Ito, M., Tsurukami, H., Nakamura, T., Doetschman, T., Coffin, JD, and Hurley, MM (2000). Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation.J. Clin. Invest. 105, 1085-1093. Hung, IH, Yu, K., Lavine, KJ, and Ornitz, DM (2007) .FGF9 regulates early hypertrophic chondrocyte differentiation and skeketal vascularization in the developing stylopod. Dev. Biol. 307, 300-313. Liu, Z., Xu, J., Colvin JS, and Ornitz, DM (2002) .Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18. Genes Dev. 16, 859-869. Ohbayashi, N., Shibayama, M., Kurotaki, Y., Imanishi, M., Fujimori T., Itoh, N., and Takada, S. (2002) .FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes Dev. 16, 870-879. Garofalo, S., Kliger-Spatz, M., Cooke, JL, Wolstin, O., Lunstrum, GP, Moshkovitz, SM, Horton, WA, and Yayon, A. (1999). Skeletal dysplasia and defective chondrocyte differentiation by targeted overexpression of fibroblast growth factor 9 in transgenic mice. J. Bone Miner. Res. 14, 1909-1915. Ornitz, DM (2000) .FGFs, heparan sulfate and FGFRs: complex interactions essential for development.BioEssays 22, 108-112. Nybakken, K., and Perrimon, N. (2002). Heparan sulfate proteoglycan modulation of developmental signaling in Drosophila. Biochim. Biophys. Acta 19, 280-291. Koziel, L., Kunath, M., Kelly, OG, and Vortkamp, A. (2004) .Ext1-dependent heparin sulfate regulates the range of Ihh signaling during endochondral ossification. Dev. Cell 6, 801-813. Plotnikov, AN, Eliseenkova, AV, Ibrahimi, OA, Shriver, Z., Sasisekharan, R., Lemmon, MA and Mohammadi, M. (2001) .Crystal structure of fibroblast growth factor 9 reveals regions implicated in dimerization and autoinhibition. Biol. Chem. 276, 4322-4329. Hecht, HJ, Adar, R., Hofmann, B., Bogin, O., Weich, H., and Yayon, A. (2001) .Structure of fibroblast growth factor 9 shows a symmetric dimer with unique receptor- and heparin- binding interfaces. Acta. Cryst. D57, 378-384. Murakami, H., Okawa, A., Yoshida, H., Nishikawa, S., Moriya, H., and Koseki, H. (2002). Elbow knee synostosis (Eks): a new mutation on mouse Chromosome 14. Mamm Genome 13, 341-344.

The first object of the present invention is to find a genetic mutation that can be involved in joint fusion and early suture fusion, and to provide a method for determining the risk of developing the disease based on the gene mutation.
The second object of the present invention is to provide a mutant FGF with modified affinity for sulfated glycosaminoglycan and tissue diffusibility.

The present inventors have identified a new missense mutation in the Fgf9 gene in which Asn143 replaces Thr in Eks mutant mice. We have named this mutant allele Fgf9 ^Eks, and this mutation prevents FGF9 homodimerization, resulting in decreased affinity of FGF9 for heparin and reduced FGFR binding and activation. I found out. At the same time, it was found that the diffusion rate was faster in tissues where FGF9 ^Eks were developing. These results strongly suggested that FGF9 ^Eks cause ectopic FGF9 signaling at the joint prosthesis site and suture line, causing elbow knee joint fusion and early suture fusion. Molecular dynamics calculations suggested that the decrease in affinity of FGF9 ^Eks for heparin was due to monomeric dominance rather than a change in its intrinsic affinity for heparin. The present inventors therefore suggested that FGF signaling is restricted through control of its affinity for HS, at least a portion of which is controlled by the monomer / dimer equilibrium of FGF9.
Based on the above findings, the present invention has been completed.

That is, the present invention relates to the following.
[1] A mutant FGF polypeptide in which Asn contributing to intermonomer hydrogen bonding in an FGF homodimer is replaced with Thr.
[2] The polypeptide according to [1], wherein FGF is FGF9.
[3] The polypeptide according to [2], wherein Asn is Asn143.
[4] The polypeptide according to [3], comprising the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 12, or 14.
[5] A polynucleotide comprising a nucleotide sequence encoding the polypeptide according to any one of [1] to [4].
[6] The polynucleotide according to [5], comprising the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 11, or 13.
[7] A partial sequence of the nucleotide sequence represented by SEQ ID NO: 1 or 3 consisting of a continuous nucleotide sequence of 15 or more bases containing cytosine of base number 428 of the nucleotide sequence represented by SEQ ID NO: 1 or 3, or its complement A polynucleotide comprising a sequence.
[8] A vector comprising the polynucleotide according to [5].
[9] A transformant introduced with the vector according to [8].
[10] A medicament comprising the polypeptide according to [1].
[11] A fracture therapeutic agent comprising the polypeptide according to [2].
[12] A method for determining the risk of developing bone fusion, comprising detecting the presence or absence of a substitution of a codon encoding Asn143 in the Fgf9 gene to a Thr-encoding codon encoding Asr143 in the FGF9 polypeptide .
[13] A risk of developing bone fusion, comprising a reagent for detecting the presence or absence of a substitution of Asn143 in Thg of FGF9 polypeptide or a codon encoding Asn143 in the Fgf9 gene to a codon encoding Thr Diagnostic agent.
[14] Testing whether or not a test substance promotes or inhibits homodimerization of FGF, and a test substance that promotes or inhibits homodimerization of FGF as a sulfated glycosamino acid of FGF A method for screening a substance capable of regulating the affinity of FGF for sulfated glycosaminoglycan, comprising obtaining the substance as a substance capable of regulating the affinity for glycan.

The present invention provides a new method for determining the risk of developing osteosynthesis. Previously, it was known that mutations in FGFR cause osteosynthesis, but the involvement of FGF mutations, which are its ligands, in osteosynthesis is not known. Therefore, the present invention provides a method for evaluating the onset risk of osteosynthesis from a completely different viewpoint.
The present invention also provides a mutant FGF having a reduced affinity for sulfated glycosaminoglycans. The mutant FGF of the present invention is excellent in diffusibility in the living body and reaches the deep part of the affected area without being trapped by the sulfated glycosaminoglycan in the vicinity of the administration site. Useful as a formulation.

1. Mutant FGF Polypeptide The present invention provides a mutant FGF polypeptide in which asparagine (Asn) contributing to intermonomer hydrogen bonding in an FGF homodimer is replaced with threonine (Thr). The mutant FGF polypeptide of the present invention includes an amino acid sequence of wild-type FGF or a partial sequence thereof, and Asn that contributes to hydrogen bonding between monomers in the FGF homodimer is substituted with Thr in the amino acid sequence. Yes.

  FGF is a known cytokine, and its amino acid sequence and nucleotide sequence are also known. The FGF used in the present invention is not particularly limited as long as it is derived from a mammal. Examples of the mammal include laboratory animals such as rodents and rabbits such as mice, rats, hamsters, and guinea pigs, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, and humans Primates such as monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees. The mammal is preferably a rodent (eg mouse) or a primate (eg human).

  FGF1 to FGF23 have been confirmed in humans and mice (Table 1). Since human FGF19 is an ortholog of mouse FGF15, the human and mouse FGF families are composed of 22 members (TRENDS in Genetics, Vol. 20, No. 11, p. 563-569, 2004, Genome Biol., vol.2, 3005.1-3005.12, 2001). In this specification, the designation of FGF follows the nomenclature of human FGF. Table 1 shows the Genbank accession numbers of the human and mouse wild-type FGF amino acid sequences.

  FGF is classified into seven subfamilies (FGF1 subfamily, FGF4 subfamily, FGF7 subfamily, FGF8 subfamily, FGF9 subfamily, FGF11 subfamily and FGF19 subfamily) based on phylogenetic analysis. The FGF1 subfamily consists of FGF1 (aFGF) and FGF2 (bFGF). The FGF4 subfamily consists of FGF4, FGF5 and FGF6. The FGF7 subfamily consists of FGF3, FGF7 (KGF), FGF10 and FGF22. The FGF8 subfamily consists of FGF8, FGF17 and FGF18. The FGF9 subfamily consists of FGF9, FGF16 and FGF20. The FGF11 subfamily consists of FGF11, FGF12, FGF13 and FGF14. The FGF19 subfamily consists of FGF19, FGF21 and FGF23.

  The FGF used in the present invention is preferably a member of the FGF9 subfamily, more preferably FGF9.

  Wild-type FGF9 polypeptide is in equilibrium between monomer and homodimer in solution. In a homodimer of FGF9 polypeptide, FGF9 monomers are associated with each other through hydrogen bonds. From the results of Examples described later, it was revealed that asparagine (Asn143) of amino acid number 143 contributes to this hydrogen bond in the FGF9 polypeptide. When the amino acid sequence of FGF is aligned based on sequence identity, Asn corresponding to Asn143 of FGF9 is highly conserved (FIG. 8), and this Asn is also hydrogen bonding between monomers for FGFs other than FGF9. The possibility of contributing to The amino acid numbers of Asn that contribute to intermonomer hydrogen bonding in each FGF are shown in Table 1.

  In this specification, the amino acid number of each FGF is expressed with reference to the full-length amino acid sequence of wild-type FGF (that is, the amino acid sequence of an immature form in which the signal peptide is not cleaved). The full-length amino acid sequence of the reference wild-type FGF is the latest version of the amino acid sequence as of March 19, 2008 among the amino acid sequences specified by the Genbank accession numbers in Table 1 above. For example, the version of Genbank accession number of the reference amino acid sequence of human FGF9 is BAA03572.1 (GI: 391719), and the version of Genbank accession number of the reference amino acid sequence of mouse FGF9 is BAA07410.1 (GI: 1107459). is there.

When the polypeptide of the present invention contains a partial amino acid sequence of FGF, and Asn contributing to hydrogen bonding between monomers in the FGF homodimer is substituted with Thr in the amino acid sequence, the partial amino acid sequence is The mutant FGF polypeptide that contains Asn that contributes to hydrogen bonding between monomers in the FGF homodimer and that is finally obtained has substantially the same activity as the polypeptide consisting of the full-length amino acid sequence of wild-type FGF As long as it has, the length is not limited. Usually, the length of the partial sequence is usually 100 amino acids or more, preferably 120 amino acids or more. Examples of “substantially the same quality of activity” include the FGF receptor activation function of a polypeptide comprising the full-length amino acid sequence of FGF. “Substantially homogeneous” means that their activities are qualitatively (eg, physiologically or pharmacologically) homogeneous. Accordingly, the quantitative factors such as the above-mentioned degree of activity are preferably equivalent, but may be different (for example, about 0.1 to about 10 times, preferably about 0.5 to about 2 times). The FGF receptor activation function is evaluated by culturing BaF3 cells expressing the corresponding FGF receptor in the presence of the mutant FGF polypeptide and measuring the degree of proliferation by incorporation of [ ³ H] thymidine. be able to. For example, the activity of a mutant FGF9 polypeptide can be assessed using BaF3 cells that expressed the FGFR2 receptor.

  Usually, the FGF polypeptide is first produced as an immature form in which a signal peptide for extracellular secretion is bound to the N-terminus. Thereafter, the signal peptide is cleaved, and immature FGF becomes mature FGF. Accordingly, a preferred embodiment of the partial amino acid sequence of FGF includes an amino acid sequence (amino acid sequence of mature FGF) obtained by removing the signal sequence from the full-length amino acid sequence of FGF. Each FGF signal peptide can be estimated from each amino acid sequence using a known sequence analysis tool such as SignalP. For example, the signal peptide of FGF9 is N-terminal 1-33 amino acids.

In the mutant FGF polypeptide of the present invention, Asr that contributes to hydrogen bonding between monomers in the FGF homodimer is replaced with Thr. For example, in the mutant FGF9 polypeptide of the present invention, Asn143 is replaced with Thr. Although this substitution causes defects in homodimerization of FGF and the affinity for sulfated glycosaminoglycans (heparin, heparan sulfate, etc.) is reduced and diffusibility in vivo is increased, The mutant FGF polypeptide has substantially the same activity as wild-type FGF. Examples of “substantially the same quality of activity” include the FGF receptor activation function of a polypeptide comprising the full-length amino acid sequence of FGF. “Substantially homogeneous” means that their activities are qualitatively (eg, physiologically or pharmacologically) homogeneous. Accordingly, the quantitative factors such as the above-mentioned degree of activity are preferably equivalent, but may be different (for example, about 0.1 to about 10 times, preferably about 0.5 to about 2 times). The FGF receptor activation function is evaluated by culturing BaF3 cells expressing the corresponding FGF receptor in the presence of the mutant FGF polypeptide and measuring the degree of proliferation by incorporation of [ ³ H] thymidine. be able to. For example, the activity of a mutant FGF9 polypeptide can be assessed using BaF3 cells that expressed the FGFR2 receptor.

In the present specification, substitution of Asn143 to Thr in FGF9 may be expressed as “N143T”. In addition, the FGF9 polypeptide in which Asn143 is replaced with Thr may be represented as “FGF9 ^Eks ”.

  In addition to the amino acid sequence derived from FGF, 1 or 2 or more (for example, about 1 to 500, preferably about 1 to 100, more preferably about 1 to 15) additions are added to the mutant FGF polypeptide of the present invention. May contain typical amino acids. Such amino acid addition is permissible as long as the mutant FGF polypeptide of the present invention has a corresponding FGF receptor activation function. The amino acid sequence to be added is not particularly limited, and examples thereof include a tag for facilitating detection and purification of fast methionine and polypeptide. Tags include Flag tag, histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (eg GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tag, etc. It can be illustrated. The position where the amino acid sequence is added is preferably the N-terminus or C-terminus of the polypeptide.

  Specific examples of the amino acid sequence contained in the polypeptide of the present invention include the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 12, or 14. The amino acid sequence represented by SEQ ID NO: 2 is obtained by replacing Asn143 of the full-length amino acid sequence of wild-type human FGF9 with Thr. The amino acid sequence represented by SEQ ID NO: 4 is obtained by replacing Asn143 of the full-length amino acid sequence of wild-type mouse FGF9 with Thr. The amino acid sequence represented by SEQ ID NO: 6 is obtained by removing the signal sequence (N-terminal 33 amino acid residues) from the amino acid sequence represented by SEQ ID NO: 2. The amino acid sequence represented by SEQ ID NO: 8 is obtained by removing the signal sequence (N-terminal 33 amino acid residues) from the amino acid sequence represented by SEQ ID NO: 4. The amino acid sequence represented by SEQ ID NO: 12 is obtained by replacing the first amino acid (Ser) of the amino acid sequence represented by SEQ ID NO: 6 with Met. The amino acid sequence represented by SEQ ID NO: 14 is obtained by replacing the first amino acid (Asn) of the amino acid sequence represented by SEQ ID NO: 8 with Met.

  The polypeptide of the present invention may be modified. Examples of such modifications include lipid chain addition (aliphatic acylation (palmitoylation, myristoylation, etc.), prenylation (farnesylation, geranylgeranylation, etc.), phosphorylation (serine residue, threonine residue, tyrosine residue) Phosphorylation), acetylation, addition of sugar chain (N-glycosylation, O-glycosylation) and the like.

  Further, in this specification, the term “polypeptide of the present invention” is used to mean a salt thereof. Polypeptide salts include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts), and particularly physiologically acceptable acid addition salts. preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The method for producing the polypeptide of the present invention is not particularly limited, and the polypeptide may be produced according to a known peptide synthesis method or may be produced using a known gene recombination technique. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. If the partial peptide or amino acid that can constitute the polypeptide of the present invention is condensed with the remaining portion, and the product has a protecting group, the protecting polypeptide can be eliminated to produce the desired polypeptide.

  When the polypeptide of the present invention is produced using gene recombination technology, first, a polynucleotide encoding the polypeptide of the present invention as described below is obtained, and a host is transformed with an expression vector containing the polynucleotide. The polypeptide can be produced by culturing the obtained transformant. The method for producing the polypeptide of the present invention using the polynucleotide and gene recombination techniques will be described later in this specification.

  The mutant FGF polypeptide of the present invention has the same physiological activity as the wild-type FGF polypeptide, and has a lower affinity for sulfated glycosaminoglycans than the wild-type FGF polypeptide, Excellent diffusibility in vivo. When the polypeptide of the present invention is administered to a living body, it can reach the deep part of the affected area without being trapped by the sulfated glycosaminoglycan in the vicinity of the administration site. Therefore, the polypeptide of the present invention is a wound, wound, burn, thrombosis, obstructive arteriosclerosis, bone disease, alopecia, liver disease, pancreatic disease, diabetes, kidney disease, heart disease, muscle atrophic lateral cord. It is useful as a therapeutic drug for sclerosis. In addition, the mutant FGF9 polypeptide of the present invention is useful as a reagent for studying the onset mechanism of osteogenesis and as a positive control in a method for determining the onset risk of osteosynthesis described below.

2. Polynucleotide The present invention provides a polynucleotide comprising a nucleotide sequence encoding the polypeptide of the present invention. The polynucleotide may be DNA, RNA, or a DNA / RNA chimera, preferably DNA. The polynucleotide may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA, or a DNA: RNA hybrid.

  Examples of the polynucleotide of the present invention include a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO: 13. The nucleotide sequence represented by SEQ ID NO: 1 is the polypeptide of the present invention comprising the amino acid sequence represented by SEQ ID NO: 2, and the nucleotide sequence represented by SEQ ID NO: 3 is the book comprising the amino acid sequence represented by SEQ ID NO: 4. The polypeptide of the present invention is represented by the nucleotide sequence represented by SEQ ID NO: 5, the polypeptide of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 6, and the nucleotide sequence represented by SEQ ID NO: 7 is represented by SEQ ID NO: 8. The polypeptide of the present invention consisting of the amino acid sequence, the nucleotide sequence represented by SEQ ID NO: 11 is the polypeptide of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 12, Each of the polypeptides of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 14 is encoded.

In the present specification, the Fgf9 gene mutation allele in which the codon encoding Asn143 is replaced with the codon encoding Thr may be represented as “Fgf9 ^Eks ” or “Eks allele”.

  For the polynucleotide of the present invention, an appropriate primer is designed by utilizing known sequence information or sequence information described in the sequence listing of the present specification, and a DNA clone or cDNA encoding FGF is used as a template. It can be amplified directly by PCR. Alternatively, a polynucleotide encoding a mutant FGF polypeptide may be synthesized by a polynucleotide synthesizer based on the sequence information.

  The obtained polynucleotide of the present invention can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. The polynucleotide may have ATG as a translation initiation codon on the 5 'end side, and may have TAA, TGA or TAG as a translation stop codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

  The polynucleotide of the present invention is useful for the production of the polypeptide of the present invention (described later) and the determination method of the present invention (described later).

  In the present invention, 15 or more bases (for example, 18 bases or more, preferably 20 bases or more, more preferably 25 bases or more) containing cytosine of nucleotide number 428 of the nucleotide sequence represented by SEQ ID NO: 1 or 3 are consecutive. A polynucleotide (partial polynucleotide of the present invention) comprising a partial sequence consisting of a nucleotide sequence or a complementary sequence thereof is provided.

  The partial polynucleotide of the present invention is useful as a probe for detecting a mutant FGF9 allele in the determination method described later.

3. Vector and transformant The present invention provides a vector comprising the polynucleotide of the present invention. Examples of the vector include an expression vector and a cloning vector, and can be selected according to the purpose. Preferably, the vector is an expression vector. The expression vector can be produced by operably linking the polynucleotide of the present invention downstream of a promoter in an appropriate expression vector. Examples of the vector include plasmid vectors and virus vectors, and can be appropriately selected depending on the host to be used.

  Examples of the host include Escherichia bacteria (Escherichia coli, etc.), Bacillus bacteria (Bacillus subtilis, etc.), yeasts (Saccharomyces cerevisiae, etc.), insect cells (night stealing) Larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), insects (larvae of silkworms), mammalian cells (rat neurons, monkey cells (COS-7, etc.), Chinese hamster cells (CHO cells, etc.) Etc.) is used.

  Examples of mammals include, for example, laboratory animals such as rodents and rabbits such as mice, rats, hamsters, and guinea pigs, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, humans, Primates such as monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees.

  Plasmid vectors derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), plasmid vectors derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmid vectors (eg, pSH19, pSH15), etc. And can be appropriately selected according to the type of host used and the purpose of use.

  The type of viral vector can be appropriately selected according to the type of host used and the purpose of use. For example, when insect cells are used as the host, baculovirus vectors can be used. When mammalian cells are used as hosts, Moloney murine leukemia virus vectors, lentivirus vectors, Sindbis virus vectors and other retrovirus vectors, adenovirus vectors, herpes virus vectors, adeno-associated virus vectors, parvovirus vectors, Vaccinia virus vectors, Sendai virus vectors, and the like can be used.

  In addition, a promoter that can initiate transcription in the host can be selected according to the type of host to be used. For example, when the host is Escherichia, trp promoter, lac promoter, T7 promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. When the host is a mammalian cell, a subgenomic (26S) promoter, CMV promoter, SRα promoter and the like are preferable.

The vector of the present invention may contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like in a functional manner, if desired. . Examples of selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (sometimes abbreviated as Amp ^r ), neomycin resistance gene (Neo). G418 resistance) which may be abbreviated as ^r ).

  The vector of the present invention is prepared according to a gene transfer method known per se (eg, lipofection method, calcium phosphate method, microinjection method, proplast fusion method, electroporation method, DEAE dextran method, gene transfer method using Gene Gun, etc.) By introducing it into a host, a transformant introduced with the vector (transformant of the present invention) can be produced. By using an expression vector as the introduced vector, the transformant can express the polypeptide of the present invention. The transformant of the present invention is useful for producing the polypeptide of the present invention.

  The polypeptide of the present invention can be produced by culturing the transformant of the present invention by a method known per se according to the type of host and isolating the polypeptide of the present invention from the culture. The transformant whose host is Escherichia is cultured in an appropriate medium such as LB medium or M9 medium, usually at about 15 to 43 ° C. for about 3 to 24 hours. The transformant whose host is Bacillus is cultured in a suitable medium, usually at about 30 to 40 ° C. for about 6 to 24 hours. The transformant whose host is yeast is cultured in a suitable medium such as a Burkholder medium, usually at about 20 ° C. to 35 ° C. for about 24 to 72 hours. Culturing of a transformant whose host is an insect cell or an insect is carried out in an appropriate medium such as Grace's Insect medium supplemented with about 10% bovine serum, usually at about 27 ° C. for about 3 to 5 days. The transformant whose host is an animal cell is cultured in an appropriate medium such as MEM medium supplemented with about 10% bovine serum, usually at about 30 ° C. to 40 ° C. for about 15 to 60 hours. In any culture, aeration and agitation may be performed as necessary. For isolation and purification of the polypeptide of the present invention from the culture, for example, the cell lysate and the culture supernatant are subjected to multiple chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography, etc. Can be achieved.

4). As pharmaceutical above, mutant FGF polypeptides of the present invention, wild-type FGF polypeptide substantially maintains the same activity, and as compared to wild-type FGF polypeptide, sulfated glycosaminoglycans It has a low affinity for and has excellent diffusibility in vivo. When the polypeptide of the present invention is administered to a living body, it can reach the deep part of the affected area without being trapped by sulfated glycosaminoglycan in the vicinity of the administration site.

  On the other hand, each FGF has wound, wound, burn, thrombosis, obstructive arteriosclerosis, bone disease, alopecia, liver disease, pancreatic disease, diabetes, kidney disease, heart disease, amyotrophic lateral sclerosis, etc. It is known or suggested to be useful in the treatment of other diseases.

  In particular, FGF2 is wound, wound, obstructive arteriosclerosis (angiogenic effect), FGF5 is obstructive arteriosclerosis (angiogenic effect), FGF9 is fracture, amyotrophic lateral sclerosis, FGF18 is fracture, hair loss It is known to be useful for the treatment of symptom (hair growth, hair growth effect). FGF5 is known to have a hair loss effect.

  Therefore, the mutant FGF polypeptide of the present invention is useful as a therapeutic agent for each of the above diseases, which has excellent permeability to the affected area.

  The pharmaceutical preparation containing the polypeptide of the present invention can be contained as the active ingredient alone or as a mixture with any other therapeutic active ingredient. In addition, these pharmaceutical preparations are produced by any method well known in the technical field of pharmaceutics by mixing the active ingredient with one or more pharmacologically acceptable carriers.

  It is desirable to use the most effective route for treatment, and it is usually administered parenterally such as transdermally, intravenously or intramedullarily.

  Formulations suitable for parenteral administration preferably comprise a sterile aqueous solution containing the active compound that is isotonic with the blood of the recipient. For example, in the case of an injection, a solution for injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of salt water and a glucose solution. To these parenteral agents, a solubilizing agent, a buffering agent, a pH adjusting agent, a tonicity agent, a soothing agent, a preservative, and the like can be further added as necessary. A preparation suitable for parenteral use may be prepared by suspending the polypeptide of the present invention in distilled water for injection or vegetable oil. In this case, a base, a suspending agent, A thickener or the like can be added. A formulation suitable for parenteral use may be a form in which the polypeptide powder or freeze-dried product of the present invention is dissolved at the time of use, and an excipient or the like can be added as necessary.

  The dosage and frequency of administration of the polypeptide of the present invention vary depending on the dosage form, patient age, body weight, nature or severity of the condition to be treated, but usually in the case of parenteral administration such as intravenous administration, About 0.001 mg to 1 g per person is administered once to several times a day. However, the dose and the number of doses vary depending on the various conditions described above.

4). Method for Determining Risk of Occlusion of Osteolysis As shown in the Examples below, an Eks mouse having a mutation of a codon encoding Asn143 to a codon encoding Thr in the Fgf9 gene is composed of a radiohumeral, a tibia and a thigh (Tibiofemoral) and skull sutures develop osteosynthesis. Therefore, the present invention relates to the risk of developing osteosynthesis comprising detecting the presence or absence of a substitution of Asn143 in Thr of FGF9 polypeptide or a codon encoding Asn143 in the Fgf9 gene to a codon encoding Thr. The determination method is provided.

  In the case of directly detecting a mutation in the polypeptide, for example, the presence of the mutant FGF9 polypeptide is immunospecifically detected by an immunological technique using an antibody that specifically recognizes the mutant FGF9 polypeptide of the present invention. Is done. Alternatively, it can be detected as a change in the mass of the polypeptide by mass spectrum.

  When using an immunological technique, for example, a sample capable of measuring a mutant FGF9 polypeptide is first prepared from a biological sample collected from a test vertebrate. As the biological sample, it is preferable to use a site capable of expressing FGF9 (bone tissue, cartilage tissue, connective tissue, kidney tissue, serum, etc.).

  When the mutant FGF9 is detected by an immunological method such as ELISA, Western blotting, spot method, aggregation method, antibody array, surface plasmon resonance, etc., a liquid sample is usually prepared from a biological sample. For example, when the biological sample is a tissue or a cell, a solubilizate is prepared by mechanically crushing them or treating them with a solubilizer. When the mutant FGF9 polypeptide of the present invention is detected by an immunological method such as flow cytometry or immunological tissue staining, biological samples such as tissues and cells are appropriately subjected to treatment such as fixation and stained with antibodies. Is done.

  As a result of the detection, when the mutant FGF9 polypeptide of the present invention is detected, it can be determined that the vertebrate individual to be measured has a high risk of developing osteosynthesis. On the contrary, when the mutant FGF9 polypeptide of the present invention is not detected, it can be determined that the vertebrate individual to be measured has a low risk of developing osteosynthesis.

  In addition, as shown in the Examples below, in Eks mice, when only one allele of the Fgf9 locus has the N143T mutation, it is compared with the case where both alleles have the N143T mutation. And the symptoms of osteosynthesis are minor. Therefore, as a result of the above detection, even when the mutant FGF9 polypeptide of the present invention is detected, when wild-type FGF9 is simultaneously detected, compared to the case where wild-type FGF9 is not detected, It can be determined that the symptoms of osteosynthesis that may develop are minor. Conversely, when wild-type FGF9 is not detected, it can be determined that the risk of developing a more severe osteosynthesis is higher than when wild-type FGF9 is detected at the same time.

  When detecting the presence or absence of a mutation of the codon encoding Asn143 in the Fgf9 gene to the codon encoding Thr, first, chromosomal DNA, total RNA, or mRNA is prepared from a biological sample collected from the test vertebrate. As the biological sample, the above-described cells, tissues, organs, and the like are used. In consideration of availability, blood, hair, nails, skin, mucous membranes, and the like are preferably used. Further, from the viewpoint of analysis of mutation, it is preferable to use a site (bone tissue, cartilage tissue, connective tissue, kidney tissue, serum, etc.) capable of expressing FGF9 polypeptide. Preparation of chromosomal DNA, total RNA, or mRNA from a biological sample can be performed by a method well known in the art, or a commercially available kit may be used.

  Determine whether the polynucleotide encoding FGF9 polypeptide contained in the chromosomal DNA, total RNA or mRNA obtained as described above has a mutation of the codon encoding Asn143 to the codon encoding Thr . The codon encoding Asn143 is usually AAT or AAC, preferably AAC. The codon encoding Thr is usually ACT, ACA, ACC or ACG, preferably ACC.

  The mutation can be analyzed by a well-known method. Examples of the method include RFLP (restriction fragment length polymorphism) method and PCR-SSCP (single-stranded DNA higher-order structure polymorphism analysis) method (see, for example, Biotechniques, 16, 296-297 (1994)). ASO (Allele Specific Oligonucleotide) hybridization method (see, for example, Clin. Chim. Acta, 189, 153-157 (1990), direct sequencing method (see, for example, Biotechniques, 11, 246-249 (1991)), ARMS ( Amplification Refracting Mutation System (for example, see Nuc. Acids. Res., 19, 3561-3567 (1991)), Denaturing Gradient Gel Electrophoresis (for example, Biotechniqus, 27, 1016-1018) (See 1999)), RNase A cleavage method (see, for example, DNA Cell. Biol., 14, 87-94 (1995)), chemical cleavage method (see, for example, Biotechniques, 21, 216-218 (1996)), DOL ( Dye-labeled Oligonucleotide Ligation (eg Genome Res., 8, 549-556 (199 8)), TaqMan PCR method (for example, see Genet. Anal., 14, 143-149 (1999)), Invader method (for example, Science, 5109, 778-783 (1993); Nat. Biotechnol., 17, 292-296 (1999)), MALDI-TOF / MS (Matrix Assisted Laser Desorption-time of Flight / Mass Spectrometry) method (see Genome Res., 7, 378-388 (1997)), TDI (Template-directed Dye) -terminator Incorporation) method (for example, see Proc. Natl. Acad. Sci. USA, 94, 10756-10761 (1997)), Southern blotting method and the like.

  For example, by using the above-described polynucleotide or partial polynucleotide of the present invention as a probe, the presence or absence of mutation can be detected by the ASO hybridization method or the RNase A cleavage method. Alternatively, in the polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 12 or 14, for SEQ ID NO: 2 or 4, the amino acid of amino acid number 143 (which is Asn or Thr) For numbers 6, 8, 12 or 14, mutations were analyzed by analyzing the nucleotide sequence of the PCR product using primers that can amplify the region containing the codon encoding amino acid number 110 (which is Asn or Thr). The presence or absence can be detected. Furthermore, the presence or absence of mutation can be detected by measuring the size of the restriction enzyme digestion product of the PCR product using a restriction enzyme site (eg, BsrI) that disappears due to the mutation.

  As a result of the detection, when the mutation is detected, it can be determined that the vertebrate individual to be measured has a high risk of developing osteosynthesis. On the other hand, when the mutation is not detected, it can be determined that the vertebrate individual to be measured has a low risk of developing osteosynthesis.

  In addition, as shown in the Examples below, in Eks mice, when only one allele of the Fgf9 locus has the N143T mutation, it is compared with the case where both alleles have the N143T mutation. And the symptoms of osteosynthesis are minor. Therefore, even if the above mutation is detected as a result of the detection, if the wild type allele (codon encoding Asn143) is detected at the same time, the wild type allele is not detected. Thus, it can be determined that the symptoms of osteogenesis that can develop are minor. Conversely, when a wild type allele is not detected, it can be determined that the risk of developing a more severe osteosynthesis is higher than when a wild type allele is detected at the same time.

  The present invention also includes a reagent for detecting the presence or absence of substitution of Asn143 in Thr of FGF9 polypeptide or mutation of a codon encoding Asn143 in the Fgf9 gene into a codon encoding Thr. Provide diagnostic agents for risk of onset. By using the diagnostic agent, it is possible to easily determine the risk of developing the above-mentioned osteosynthesis.

Although it does not specifically limit as this reagent, For example,
(1) an antibody that specifically recognizes the mutant FGF9 polypeptide of the present invention;
(2) the polynucleotide of the present invention;
(3) the partial polynucleotide of the present invention;
(4) In the polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 12 or 14, SEQ ID NO: 2 or 4 is the amino acid of amino acid number 143 (the amino acid is Asn or Thr) A primer that can amplify a region comprising a codon that encodes the amino acid of amino acid number 110 for SEQ ID NO: 6, 8, 12, or 14 (the amino acid is Asn or Thr);
Etc. can be mentioned.

  In addition to the reagents described above, various reagents used in the above-described testing methods (eg, protein extraction reagents, labeled antibodies, chromosomal DNA preparation reagents, total RNA preparation reagents, mRNA preparation reagents, restriction enzymes (eg, BsrI), reagents) And a buffer for diluting a biological sample, a positive control, a negative control, a reaction container, an instruction describing a test protocol, and the like) may be used as a kit for determining the risk of developing bone fusion. These elements can be mixed in advance if necessary. Moreover, a preservative and preservative can also be added to each element as needed. The determination kit is extremely useful because it makes it possible to easily determine the risk of developing the above osteosynthesis.

5). Screening Method As shown in the examples described later, when the homodimerization of FGF is inhibited and the amount of monomer increases, the affinity of FGF for sulfated glycosaminoglycans decreases. Therefore, by controlling the homodimerization of FGF, the affinity of FGF for sulfated glycosaminoglycan can be adjusted. Accordingly, the present invention is to test whether or not a test substance promotes or inhibits homodimerization of FGF, and to test the test substance that promotes or inhibits homodimerization of FGF for sulfation of FGF. Provided is a screening method for a substance capable of regulating the affinity of FGF for sulfated glycosaminoglycan, comprising obtaining the substance as a substance capable of regulating the affinity for glycosaminoglycan.

  The test substance used for the screening method of the present invention may be any known compound and novel compound, for example, using nucleic acid, carbohydrate, lipid, protein, peptide, low-molecular-weight organic compound, combinatorial chemistry technique. Examples include a compound library prepared, a random peptide library prepared by solid-phase synthesis or a phage display method, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.

  In testing whether or not a test substance promotes or inhibits homodimerization of FGF, FGF is brought into contact with the test substance in an appropriate buffer, and the homodimer of FGF in the presence of the test substance. Measure the degree of merification. The degree of homodimerization can be measured by a known method such as analytical ultracentrifugation or gel filtration chromatography. For example, analytical ultracentrifugation can measure the average molecular weight of FGF in solution. When all the FGF in the solution is monomer, this average molecular weight is the same as the calculated molecular weight of the FGF monomer. On the other hand, when all FGF in the solution is a dimer, the average molecular weight is twice the calculated molecular weight of the FGF monomer. And when a monomer and a dimer coexist, the average molecular weight shows a value between 1 and 2 times the calculated molecular weight of the FGF monomer, and when the dimerization is promoted, the average molecular weight Therefore, the monomer / dimer ratio can be calculated using this average molecular weight as an index.

  Next, the degree of homodimerization of FGF in the presence of the test substance is compared with the degree of homodimerization of FGF in the absence of the test substance. The comparison of the degree of homodimerization is preferably performed based on the presence or absence of a significant difference. The degree of homodimerization in the absence of the test substance was measured at the same time, even if it was a value measured in advance for the measurement of the degree of homodimerization in the presence of the test substance. Although it may be a value, it is preferably a value measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

  As a result of the comparison, a test substance that promotes or inhibits homodimerization of FGF is selected, and the substance is obtained as a substance that can regulate the affinity of FGF to sulfated glycosaminoglycan. That is, the test substance that promoted homodimerization of FGF was obtained as a substance that enhances the affinity of FGF for sulfated glycosaminoglycan, and the test substance that inhibited homodimerization of FGF was Acquired as a substance that reduces the affinity of FGF for sulfated glycosaminoglycans.

  When the substance obtained by the above screening method is used in combination with a drug containing FGF, the diffusibility of FGF in vivo can be controlled. For example, when FGF is desired to be localized at the administration site, a substance that enhances the affinity of FGF for sulfated glycosaminoglycan may be used in combination with FGF. On the other hand, when FGF is desired to reach the deep part of the affected area, a substance that lowers the affinity of FGF for sulfated glycosaminoglycan may be used in combination with FGF.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

(Method)
Detection of mutations in the Fgf9 gene To identify mutations that contribute to the Eks mutant phenotype, reverse transcription of mRNA—PCR (RT-PCR) and direct sequencing of RT-PCR products (ABI PRIZM 310 Genetic Analyzer; Applied Biosystems Foster City, Calif.), The sequences of cDNA from normal mice (+ / +), heterozygous mice (Eks / +) and homozygous mice (Eks / Eks) were examined. For genotyping of the Eks allele, use the specific primers (5′-CACAGGAATGTGTGTTCAGA-3 ′ (SEQ ID NO: 9) and 5′-GGTCCACTGGTCTAGGTAAA-3 ′ (SEQ ID NO: 10)) PCR product was subsequently digested with BsrI restriction enzyme. Wild type mice show two bands of 147 bp and 42 bp, while the Eks allele shows one band of 189 bp.

Skeletal and histological specimen preparation E17.5 fetal bone and cartilage were stained with alizarin red and alcian blue as described previously (Kessel and Gruss. 1990). For histological preparation, tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sliced to 5 μm, and stained with hematoxylin and eosin.

As described in in situ hybridization (Kessell and Gruss. 1991), radiolabeled Fgf9 (MGI: 104723), Gdf5 (MGI: 95688), Col2a1 (MGI: 88452), Spp1 (MGI: 98389), Runx2 (MGI: 99829), Fgfr1 (MGI: 95522), Fgfr2 (MGI: 95523) and Fgfr3 (MGI: 95524) RNA were used for in situ hybridization of paraffin sections. Whole mount in situ hybridization was performed as previously described (Iseki et al., 1997).

Analytical ultracentrifugation All analytical ultracentrifugation was performed using a Beckman Coulter XL-I analytical ultracentrifuge. The sample was dissolved in 25 mM ammonium acetate buffer (pH 5.5) containing 120 mM NaCl. The specific volume was estimated to be 0.7317 mL / g (normal FGF9 protein; FGF9 ^WT ) or 0.7322 mL / g (Eks mutant FGF9 protein; FGF9 ^Eks ) by SEDNTERP software. All experiments were performed at 20 ° C. and an absorption wavelength of 280 nm. Sedimentation equilibrium tests were conducted at 0.8, 0.4 and 0.2 mg / ml with a 6 channel centerpiece. Data were acquired at 12, 14 and 16 krpm for FGF9 ^WT and 14, 16 and 18 krpm for FGF9 ^Eks . A total equilibration time of 16 hours was used to scan for each speed at 12, 14 and 16 hours. Sedimentation equilibrium data was analyzed using Beckman XL-A / XL-I data analysis software. The sedimentation rate test was conducted with a double sector centerpiece. The protein concentration was 0.4, 0.3 or 0.2 mg / ml. Absorbance data was scanned 100 times every 5 minutes at 40 krpm. The measurement data was analyzed with SEDFIT software.

Mitogenic assay The ability of FGF9 ^WT and FGF9 ^Eks to transmit signals via FGFR was determined by mitogenic assay using BaF3 cells expressing specific FGFR as described (Ornitz et al., 1996). Was analyzed. 5000 cells per well of a 96 well assay plate were seeded into medium containing various concentrations of FGF9 and heparin (Wako). Heparin and FGF9 ^WT or FGF9 ^Eks were added to each well to make a total volume of 200 μl per well. The cells were then incubated for 36 hours at 37 ° C. 1 μCi of [ ³ H] thymidine dissolved in 20 μl of medium was added to each well. After 4 hours, cells were harvested by filtration through glass fiber paper and incorporated [ ³ H] thymidine was counted with a Wallac MicroBeta TriLux scintillation counter (PerkinElmer).

Heparin affinity chromatography 3 mg of FGF9 ^WT and FGF9 ^Eks were loaded onto a 1 ml HiTrap heparin column (GE healthcare) equilibrated with 25 mM ammonium acetate buffer (pH 5.5) containing 120 mM NaCl. Bound FGF9 ^WT and FGF9 ^Eks were eluted with a linear gradient of NaCl (120 mM-2.0 M) in the same buffer.

Surface Plasmon Resonance Analysis of FGF9- Heparin Interactions Surface plasmon resonance analysis to measure FGF9 ^WT -heparin and FGF9 ^Eks -heparin interactions was performed using a BIAcore 3000 instrument (Biacore AB, Uppsala, Sweden). In order to immobilize heparin (Wako) on the streptavidin-coupled sensor chip SA, 100 μg / ml biotinylated heparin in HBS-EP buffer was injected at a flow rate of 10 μl / min, and 63 reaction units (RU) were immobilized. Phased. All tests were performed at room temperature and refractive index anomalies due to bulk solvent effects were corrected by subtracting the response on the uncoated sensor chip at the FGF9 ^WT and FGF9 ^Eks concentrations used. To obtain kinetic data, different concentrations of ^analytes (FGF9 ^WT and FGF9 ^Eks ) in HBS-EP were injected onto the heparin sensor chip at a flow rate of 20 μl / min. At the end of each sample injection (120s), HBS-EP buffer was passed over the sensor surface and the dissociation phase was monitored. Following dissociation of 120 s, the sensor surface was completely regenerated by injection of 5 μl of 1M NaCl (in HBS-EP). Five different analyte concentrations were used to determine the kinetic parameters for each interaction. Kinetic parameters were obtained by fitting the sensorgrams comprehensively to a 1: 1 model using BIAevaluation software.

Molecular dynamics (MD) simulations monomer FGF9 ^WT, dimer FGF9 ^WT, monomer FGF9 ^WT - heparin, dimeric FGF9 ^WT - start to become structures for these MD for heparin, and ^{FGF9 Eks} is , PDB (PDB ID: 1IHK) (Plotnikov et al, 2001). The structures of monomeric FGF9 ^WT and dimeric FGF9 ^Eks were constructed based on FGF9 ^WT using molecular modeling software MOE (Chemical Computing Group, Inc.). Hexasaccharide (UAP-SGN-IDU-SGN-IDU-SGN) was used as the heparin oligosaccharide. UAP is 1,4-dideoxy-5-dehydro-O2-sulfo-glucuronic acid, SGN is O6-disulfo-glucosamine, and IDU is 1,4-dideoxy-O2-sulfo-glucuronic acid. For the simulation of monomeric FGF9 ^WT -heparin and dimeric FGF9 ^WT -heparin, the molecular docking program GOLD (version 3.0) (Verdonk et al, 2003) was used to convert heparin oligosaccharides to the FGF9 ^WT structure obtained from MD simulation. Combined. In the docking protocol, standard GA parameter default settings were used and GoldScore was used as the scoring function. The structures of monomeric FGF9 ^Eks -heparin and dimeric FGF9 ^Eks -heparin were constructed in the same manner as the FGF9 ^WT -heparin complex. The starting structure for MD simulation was enclosed in a sphere with TIP3P water molecules (Jorgensen et al., 1983). After energy minimization, all MD simulations were performed at 300 K for 10 ns using a modified Amber 8.0 (Case et al., 2004) for the MDGRAPE3 system (Narumi et al., 2006). Amber ff03 force field (Duan et al., 2003) was applied and the simulation time step was set to 1 fs. The binding free energy was calculated by the molecular functional Poisson-Boltzmann / surface area (MM-PBSA) method (Srinivasan et al., 1998) using the final 2 ns MD orbital.

Retroviral ectopic expression mouse FGF9 ^WT and FGF9 ^Eks cDNA were cloned into RCASBP (A) vector (Hughes et al., 1987). The virus solution was injected into the hindlimb buds of a Hamburger-Hamilton (HH) stage 17 chicken embryo. Mouse Fgf9 transcript expression and skeletal changes were examined 2 and 5 days after injection, respectively.

Subcutaneous insertion of FGF9 ^Eks beads in mouse fetal skulls AffiGel-Blue beads (Bio-Rad, Richmond, Calif.) Immersed in 100 μg / ml FGF9 ^WT or FGF9 ^Eks were described on E15 mouse skulls by extrauterine surgery. (Iseki et al., 1997). Surgery heads were collected 24 hours later and Spp1 transcripts were detected by whole mount in situ hybridization. The Spp1 expression area was measured using NIH image software.

Implantation of FGF9 ^Eks beads into mouse forelimb buds AffiGel-Blue beads (Bio-Rad, Richmond, Calif.) Soaked in 500 μg / ml FGF9 ^WT or FGF9 ^Eks were used for E10.5 Fgf9 ^{− / −} Transplanted into the dorsal and central regions. The limb buds were then transwelled in a serum-free medium [BGJb, 2 mg / ml BSA, penicillin (50 units / ml), streptomycin (50 μg / ml)] in a humid, 37 ° C. and 5% CO ₂ environment. Incubate on filter (Costar, Coaning) for 2 hours. Explants were fixed in 4% paraformaldehyde and embedded in paraffin. Sections through the equator of the beads were analyzed for exogenous FGF9 using an anti-human FGF9 antibody (R & D Systems) and a cell and tissue staining kit HRP-AEC system (R & D Systems). Signal area and intensity were analyzed using NIH image software.

FGF9 ^WT and FGF9 ^Eks expression and purified purified FGF9 ^WT and FGF9 ^Eks Except for the production of recombinant protein in E. coli, it was produced essentially according to the method described previously (Koyama et al., 2001). Since the first 33 N-terminal residues are only involved in secretion, we have the cDNA encoding the first Met followed by the 35th to 208th polypeptide sequences of FGF9 ^WT and FGF9 ^Eks. Fragments were produced by RT-PCR. Each fragment was inserted between the NdeI and HindIII sites in the pColdIV bacterial expression vector (Takara). The resulting plasmid was transformed into E. coli. E. coli strain BL21 was introduced and recombinant protein production was induced in SB medium supplemented with 1 mM isopropyl-1-thio-β-D-galactopyranoside at 15 ° C. for 24 hours. Bacteria were collected and subsequently disrupted by lysosome treatment and sonication. Cell debris was removed by centrifugation and FGF9 ^WT and FGF9 ^Eks were recovered from the supernatant by fractionating with 25% saturation of ammonium sulfate followed by sedimentation of the supernatant with 50% ammonium sulfate (FIG. 9A). The precipitate was dissolved in 25 mM ammonium acetate buffer (pH 5.5) and dialyzed against 25 mM ammonium acetate buffer (pH 5.5) containing 1% ammonium sulfate. As shown in FIG. 14, the obtained fractions were subjected to a series of chromatography columns (TOYOPEARL AF-Heparin HC-650M column (TOSOH), TOYOPEARL CM-650M column (TOSOH), TOYOPEARL HW-50F column (TOSOH)). Further fractionation by passing through. The inventors performed these chromatographic steps using the buffer system described previously (Koyama et al., 2001). In this way, 20 L of E. 200 mg of highly purified FGF9 ^WT and 100 mg of FGF9 ^Eks were obtained from the ^E. coli culture (FIG. 9B).

Analytical gel filtration chromatography Purified FGF9 ^WT and FGF9 ^Eks (2 mg / ml in 100 μl) equilibrated with 25 mM ammonium acetate buffer (pH 5.5) containing 120 mM NaCl (GE healthcare) ). Samples were eluted with the same buffer.

(result)
The Eks mutation caused by a missense mutation in Fgf9 was mapped between polymorphic markers D14Mit62 and D14Mit5 on mouse chromosome 14 (Murakami et al., 2002). Of the 169 genes located in this section, the focus was on Fgf9. As a result of sequence analysis of Fgf9 cDNA from homozygous Eks mice, it was found that a substitution from A to C occurred at position 428, resulting in a substitution of Asn143 to Thr (FIG. 1A). Interestingly, the Asn143 residue is highly conserved in most FGF proteins (Figure 8).

We used a genetic approach to determine whether the N143T substitution in Fgf9 contributes to the Eks phenotype. Since the BsrI restriction enzyme site present in the C57BL / 6, DBA / 2J and JF1 / Ms strains is removed by the Fgf9 ^Eks mutation, the wild type and the mutant allele can be distinguished by digesting the PCR fragment with BsrI ( FIG. 1B). To test whether the Fgf9 ^Eks mutation and Eks locus could be genetically isolated, Fgf9 ^{Eks / +} mice were cross-fertilized. Of the 976 offspring or fetuses tested, 230 (24%) and 516 (53%) have homozygous and heterozygous Eks phenotypes, respectively, and are homozygous for the N143T mutation, respectively. And heterozygous. Therefore, the Eks phenotype and the mutation in Fgf9 were co-segregated in all cases.

We next tested whether the Eks phenotype could be altered by the introduction of the Fgf9 null mutation. Homozygous Fgf9 null mutant (Fgf9 ^{− / −} ) exhibits pulmonary hypoplasia, male-to-female reversal, and rhizomelia, but does not show joint or suture osteosynthesis ( Colvin et al, 2001; Colvin et al., 2001; Hung et al., 2007). We compared the phenotype of the formulated heterozygous mutant (Fgf9 ^{Eks / −} ) to Eks homozygous mice (Fgf9 ^{Eks / Eks} ) and Fgf9 ^{− / −} mice. The slight pulmonary dysplasia seen in Fgf9 ^+/− and Fgf9 ^{Eks / +} mice was significantly worse in Fgf9 ^{Eks / −} mice (FIG. 1C). The extent of pulmonary dysplasia in Fgf9 ^{Eks / −} mice was intermediate between Fgf9 ^{− / −} and Fgf9 ^{Eks / Eks} . Elbow osteosynthesis in Fgf9 ^{Eks / −} mice was accompanied by ossification of cartilage linked as seen in Fgf9 ^{Eks / Eks} mice, but Fgf9 ^{Eks / +} , Fgf9 ^{− / −} and Fgf9 ^+/−. It was not observed in mice (FIG. 1D). Fgf9 ^{Eks / −} mice showed no sex change. The absence of recombination between Eks and Fgf9 in nearly 2000 meiosis and the enhanced lung and joint phenotype in Fgf9 ^{Eks / −} mice compared to heterozygotes indicate that the Eks mutation is Fgf9. Provides strong evidence of being an allele of

Similar to the phenotype of Fgf9 ^{Eks / Eks} and gain-of-function Fgfr mutants . We firstly compared the Eks mutant with Fgfr1 (Wang et al., 2001) and Fgfr2c (Fgfr2 ^C342Y ) (Eswarakumar et al. We investigated whether there were phenotypic similarities among the gain-of-function mutants of 2004). In Fgfr1 gain-of-function transgenic mice, a failure first occurs in the initiation of elbow joint development (Wang et al., 2001), so we examined joint development in Fgf9 ^{Eks / Eks} mice (FIG. 2A). . Growth and differentiation factor 5 (Gdf5) (Storm and Kingsley, 1996) and procollagen, type II alpha 1 (Col2a1) (Nalin et al., 1995) were specifically expressed in the elbow joint planned area and cartilage aggregation, respectively. . Gdf5 expression in the planned joint space was observed at E11.5 in Fgf9 ^{+ / +} control mice (FIGS. 2Aa and 2Ae), but none in Fgf9 ^{Eks / Eks} mice (FIG. 2Af). Analysis of cartilage revealed a loss of Col2a1 expression in the planned area of the elbow joint in the E11.5 wild-type fetus (FIG. 2Ag). Consistent with the deletion of Gdf5 expression, there was no deletion site for Col2a1 expression in Fgf9 ^{Eks / Eks} mice (FIG. 2Ah). This molecular phenotype is very similar to that seen in transgenic mice ectopically expressing the active Fgfr1 kinase domain in the planned joint area (Wang et al., 2001).

Early fusion of the skull suture in Fgfr2 ^C342Y mice is the result of excessive osteogenic differentiation within the skull suture mesenchyme (Eswarakumar et al., 2004). To elucidate whether Fgf9 ^{Eks / Eks} mice have similar histological characteristics, we have identified an early osteoblast differentiation marker, Secreted phosphoprotein 1 (Spp1, also known as Osteopontin in developing skull sutures). ) (Iseki et al., 1997) and Runt related transcription factor 2 (Runx2) (Yoshida et al., 2005) were examined. At E15.5, Fgf9 ^{+ / +} and Fgf9 ^{Eks / Eks} mice showed similar skull suture histology (FIGS. 2Ba-2Bf). However, at E16.5, in the Fgf9 ^{Eks / Eks} mice, the overlap of the frontal and parietal bones was greater compared to Fgf9 ^{+ / +} (FIGS. 2Bg and 2Bh). The expression domain of Sppl, a differentiation marker for preosteoblasts and osteoblasts, showed a wide separation of frontal and parietal bone in Fgf9 ^{+ / +} mice (FIG. 2Bi), however, in Fgf9 ^{Eks / Eks} mice There was a significant overlap (FIG. 2Bj). Runx2 is expressed in immature osteoblasts at the leading edge of the frontal and parietal bones. Runx2 expression filled sutures in both E15.5 and E16.5 Fgf9 ^{Eks / Eks} mice, suggesting the accumulation of immature osteoblasts within the cranial suture mesenchyme (FIGS. 2Bf and 2Bl). Furthermore, the strength of the Runx2 expression domain appears to be low in Fgf9 ^{Eks / Eks} mice (FIG. 2B1), suggesting that osteoblast differentiation is enhanced. Taken together, these findings suggest that the Fgf9 ^Eks mutation mediates excessive FGFR signaling in future joints and sutures, and consequently inhibits initiation of joint and suture development.

Eks mutation impairs homodimerization of FGF9 The extent of dimerization of FGF9 ^WT and FGF9 ^Eks proteins was compared by analytical ultracentrifugation. FGF9 ^WT and FGF9 ^Eks It was expressed in E. coli and purified by column chromatography (FIG. 9).

The molecular weight and equilibrium constant of the purified recombinant proteins FGF9 ^WT and FGF9 ^Eks were determined by sedimentation equilibrium centrifugation (FIGS. 3A and 3B). The measured average molecular weights of FGF9 ^WT and FGF9 ^Eks were 39,264 and 32,929 Da, respectively, whereas the calculated molecular weights of the monomers were 20,090 and 20,077 Da, respectively. . These data suggest that FGF9 ^Eks exist in equilibrium as both monomers and dimers, whereas FGF9 ^WT exists mostly as dimers. FGF9 ^WT and ^FGF9 binding constants on calculations ^Eks were respectively 10.4MyuM ^-1 and 0.119μM ^-1. These results are consistent with the fact that FGF9 ^Eks elutes later than FGF9 ^WT in the gel filtration column (FIG. 10). The inventors further measured the sedimentation coefficient of FGF9 ^Eks by sedimentation rate centrifugation. The overlay plot of the c (s) sedimentation coefficient distribution shows that FGF9 ^WT has a unimodal peak at 3.0S, whereas FGF9 ^Eks has bimodal peaks (2.2S and 3.1S) (Fig. 3C and 3D). This suggests that FGF9 ^Eks exists mainly as a monomer, whereas FGF9 ^WT exists mainly as a dimer. Therefore, FGF9 ^Eks are impaired in homodimer formation, which may affect the self-inhibitory function that controls the activity of FGF9, which is inferred from structural analysis.

FGF9 ^Eks do not mediate excessive signaling through FGFRs We next mitogenic FGF9 ^WT and FGF9 ^Eks against BaF3 cells expressing individual receptors (Ornitz et al., 1996) By comparing the activity, the FGFR activating ability of FGF9 ^WT and FGF9 ^Eks was compared. BaF3 cell lines expressing FGFR were treated with various concentrations of FGF9 in the presence of 1 μg / ml heparin. Compared to FGF9 ^WT , FGF9 ^Eks showed less activity against cells expressing FGFR1c, -2b, -2c, -3b and -4 (Table 2, FIGS. 11A-G).

Table 2 shows a comparison of the mitogenic activity of FGF9 ^Eks with that of FGF9 ^WT . The mitogenic activity of FGF9 ^Eks was averaged at two concentrations of 200 and 1000 pM FGF9 for FGF9 concentration dependent assays and 1.56 and 6.25 μg / ml for heparin concentration dependent assays. Normalized to activity of FGF9 ^WT at concentration.

To examine the ability of heparin to enhance FGF9 activity, the BaF3 cell line was treated with various concentrations of heparin in the presence of 0.2 nM FGF9 ^WT or FGF9 ^Eks . FGF9 ^Eks did not show a heparin concentration-dependent mitogenic response in some FGFRs (Table 2, FIGS. 11H-11N). Since FGF9 ^Eks did not show excessive signaling via FGFR, other properties of the mutant protein were thought to contribute to the Eks mouse phenotype.

Asn143Thr mutation in FGF9 reduces affinity for heparin Reduced heparin concentration-dependent mitogenic activity of FGF9 ^Eks suggests that FGF9's affinity for heparin may be reduced. To investigate this possibility, the inventors first measured FGF9 / heparin affinity by heparin affinity chromatography. FGF9 ^WT was 1.50 M NaCl and eluted from heparin-conjugated agarose as a single peak (FIG. 4A). In contrast, most FGF9 ^Eks were eluted with 1.38 M NaCl and a small fraction was eluted with 1.10 M NaCl. Furthermore, the elution profile of FGF9 ^Eks was broader than that of FGF9 ^WT .

We next measured the rate constant of the FGF9 ^Eks / heparin interaction using surface plasmon resonance analysis (FIGS. 4B and 4C). The resulting sensorgrams were comprehensively fitted to a 1: 1 interaction model to determine kinetic parameters (Table 3).

Table 3 shows the kinetic parameters of the interaction of FGF9 ^WT and FGF9 ^Eks with heparin. In three independent _experiments, it was calculated from k a, sensorgram using five concentrations of _{k b} and analyte _{K D} values (FGF9 ^WT and ^{FGF9 Eks).}

FGF9 ^Eks binding rate constant _{(k a)} is, while was slightly larger than that of FGF9 ^{WT, FGF9 Eks} dissociation rate constant _{(k b)} is was 18 times greater than that of FGF9 ^WT. The dissociation constants (K _D ) of FGF9 ^WT and FGF9 ^Eks were 0.71 ± 0.02 nM and 5.24 ± 0.03 nM, respectively, indicating that the heparin affinity of the FGF9 ^Eks protein was reduced by 86%. means.

Monomeric FGF9 has a lower affinity for heparin than dimeric FGF9 The FGF9 ^Eks mutation affects the affinity for heparin / HS as well as the monomer / dimer equilibrium. It is unclear whether the Asn143Thr mutation directly affected the affinity of FGF9 for heparin or directly affected homodimerization and secondarily affected heparin affinity is there. Since the monomeric and dimeric forms of FGF9 are in equilibrium, it is not possible to directly biochemically measure the affinity of the two FGF9s for heparin. We therefore analyzed the configuration of the heparin binding domain in FGF9 monomers and dimers using molecular dynamics (MD) simulations and the binding between FGF9 and heparin using the MM-PBSA method. Free energy was calculated.

MD simulations for the FGF9 ^WT dimer and the FGF9 ^Eks dimer showed that the Eks mutation resulted in the loss of two hydrogen bonds between the two FGF9 monomers. Asn143 formed intermonomer hydrogen bonds with Tyr67, Arg69, and Tyr145 in the dimeric FGF9 ^WT (FIG. 5A), whereas Thr143 formed hydrogen bonds only with Tyr145 in the dimeric FGF9 ^Eks (FIG. 5B). . Other intermonomer hydrogen bonds, Arg62-Asp193 and Arg64-Asp193, were not affected by the Eks mutation. As a result, the binding free energy of homodimerization for FGF9 ^WT and FGF9 ^Eks was −101.21 and −69.47 kcal / mol, respectively. From this difference, FGF9 ^Eks is expected to shift the monomer / dimer equilibrium to the monomer side compared to FGF9 ^WT . This expectation is in good agreement with the test data shown in FIG.

To model the heparin binding affinity of FGF9, we have a 2: 2: 2 FGF2-FGFR1-heparin crystal structure (Protein Data Bank (PDB) ID: 1FQ9) (Schlessinger et al., 2000). Based on the above, MD simulations of 2: 2 FGF9 ^WT -heparin and 2: 2 FGF9 ^Eks -heparin complex were performed. Since the structure of the two heparin oligosaccharides in each complex was affected by strong electrostatic repulsion, one heparin oligosaccharide was ejected from the complex (data not shown). This analysis suggested that the 2: 2 FGF9-heparin complex is unstable. In contrast, MD simulations of 2: 1 FGF9 ^WT -heparin, 2: 1 FGF9 ^Eks -heparin, 1: 1 FGF9 ^WT -heparin and 1: 1 FGF9 ^Eks -heparin complex are stable in these complexes. I suggested that. MD simulation of FGF9-heparin complex shows heparin binding freedom between 2: 1 FGF9 ^WT -heparin (dimeric FGF9 ^WT -heparin) and 2: 1 FGF9 ^Eks -heparin (dimeric FGF9 ^Eks -heparin) complex. No energy difference was shown (FIGS. 5C and 5D). This is a strong interaction between negatively charged heparin oligosaccharide chains and the sequence of basic amino acid residues located at the heparin binding site in the groove of the dimer interface in both dimer complexes Is due to. Furthermore, the mobility of the heparin oligosaccharide chains promotes the maintenance of electrostatic interactions. Similarly, there was no significant difference in the heparin binding free energy of 1: 1 FGF9 ^WT -heparin (monomer FGF9 ^WT -heparin) and 1: 1 FGF9 ^Eks -heparin (monomer FGF9 ^Eks -heparin) complex. (FIGS. 5E and 5F). This is also due to the mobility of the heparin oligosaccharide chain, the strong negative charge of the heparin oligosaccharide chain, and the presence of many basic amino acid residues at the heparin binding site. Thus, the Eks mutation does not significantly affect the heparin binding affinity of the monomeric or dimeric FGF9-heparin complex. Since the free energy of heparin binding of dimeric FGF9 (FIGS. 5C and 5D) is smaller than that of monomeric FGF9-heparin of both FGF9 ^WT and FGF9 ^Eks (FIGS. 5E and 5F), the affinity of FGF9 ^Eks protein for heparin The decrease is strongly suggested mainly due to the shift of the monomer / dimer equilibrium to the monomer side.

Expression of Fgf9 and Fgfr in future elbow joints and cranial sutures We have found that FGF9 ^Eks has a low affinity for heparin, thus increasing the diffusivity of FGF9 ^Eks within the tissue and outside the normal signaling domain It was hypothesized that it would cause ectopic localization, resulting in ectopic activation of FGFRs.

We examined the expression of Fgf9 and Fgfr1, -2 and -3 in the forelimb buds of E10.5 and E11.5 mice. Fgf9 was predominantly expressed in myoblasts in both Fgf9 ^{+ / +} and Fgf9 ^{Eks / Eks} (FIGS. 12A, 12B, 12I, and 12J). In E10.5, in both Fgf9 ^{+ / +} and Fgf9 ^{Eks / Eks} tissues, Fgfr1, -2 and -3 were expressed dispersedly in the limb bud mesenchyme and overlapped with the expression domain of Col2a1 ( FIG. 12C-12H). In E11.5, Fgfr2 and Fgfr3 were mainly expressed in the cartilage aggregation site, and Fgfr1 was expressed so as to surround the cartilage aggregation site (FIGS. 12K-12P). Therefore, Fgfrs is expressed in the mesenchymal cells of the planned elbow joint area.

  Previous reports have shown that Fgf9 is expressed in the developing frontal and parietal bones, particularly strongly in the periphery of the bone (Hajihosseini and Heath, 2002). Fgfr1, -2 and -3 are expressed in and around the developing frontal and parietal bone domains (Johnson et al., 2000). Therefore, the expression patterns of Fgf9 and Fgfrs are consistent with our fusion onset mechanism model.

The ectopic expression of FGF9 ^WT and FGF9 ^Eks can both inhibit joint and suture development. We next introduced FGF9 ^WT and FGF9 ^Eks into chicken limb buds by retroviral (RCAS) transduction. Was ^ectopically expressed to examine the inhibitory effect of FGF9 ^WT and FGF9 ^Eks on joint development. RCAS-FGF9 ^WT , RCAS-FGF9 ^Eks , or empty RCAS virus was used to infect the future hindlimb bud region of the lateral plate mesoderm. Exogenous FGF9 ^WT and FGF9 ^Eks were expressed throughout hindlimb buds 2 days after infection (FIGS. 6Aa and 6Ab). When empty virus was used, no abnormalities were observed (FIG. 6Ac-6Ae), but ectopic expression of FGF9 ^WT caused significant hindlimb joint fusion and skeletal atrophy (FIG. 6Ac). Skeletal atrophy was similar to that observed in transgenic mice overexpressed Fgf9 under the Col2a1 promoter and enhancer (Garofalo et al., 1999). Interestingly, when RCAS-Fgf9 ^Eks virus was used for infection, knee joint formation was reproducibly impaired, but no hindlimb atrophy was observed (FIG. 6Ad). This difference between FGF9 ^WT and FGF9 ^Eks may be due to weak FGF9 ^Eks signaling through some FGFR. Defects in arthroplasty are also found in the distal joints of the hind limb (FIG. 13), a phenotype also found in Fgfr1 gain-of-function transgenic mice (Wang et al., 2001).

To examine the inhibitory effect of FGF9 ^WT and FGF9 ^Eks on suture development, FGF9-immersed Affigel-Blue beads were implanted by extrauterine surgery into E15 normal mouse fetal skull sutures. Prior to transplantation, the inventors examined the amounts of FGF9 ^WT and FGF9 ^Eks contained in one Affigel-Blue bead and confirmed that approximately equal amounts were included (FIG. 14). The expression of Spp1, an early osteoblast differentiation marker whose expression is induced by FGF9, was examined 24 hours after in utero incubation. In normal mouse fetuses, Spp1 expression was not observed at the suture site between the frontal bone primordium and the parietal bone primordium, but when FGF9 ^WT and FGF9 ^Eks beads were implanted, Spp1 expression was observed at both suture sites. (FIGS. 6Ba and 6Bc).

FGF9 ^Eks are more diffuse than FGF9 ^WT in developing tissues
To further verify the hypothesis that increased tissue diffusivity of FGF9 ^Eks could explain the joint fusion phenotype in Eks mice , diffusivity of FGF9 ^WT and FGF9 ^Eks was measured in the skull after bead transplantation (FIG. 6B). Since FGF9 induces Spp1 expression, the present inventors measured the high expression region of Spp1 as an index of the distance that FGF9 exerts its effect. Transplantation of FGF9 ^Eks beads expands the region in which Spp1 is induced to expression compared to transplantation of FGF9 ^WT beads, suggesting that FGF9 ^Eks protein can diffuse over a wider region.

Next, we examined the diffusivity of FGF9 ^{Eks in} the forelimb buds. FGF9 ^WT or FGF9 ^Eks soaked Affigel-Blue beads were implanted into the dorsal and central forelimb bud regions of E10.5 Fgf9 ^{− / −} fetuses. Two hours after transplantation, the tissue was fixed and embedded, a section was prepared at the equator of the bead, and FGF9 ^WT or FGF9 ^Eks protein released from the bead into the mesenchymal tissue was subjected to immunohistochemistry using an FGF9 antibody. (Figures 6Ca and 6Cb). FGF9 ^Eks penetrated the limb bud mesenchyme to a greater extent than FGF9 ^WT (FIG. 6Cc), supporting the hypothesis that FGF9 ^Eks is more diffusive than FGF9 ^WT . In summary, our results indicate that joint formation inhibition and early suture fusion in Eks mutant mice were caused by ectopic FGF9 signaling through high interstitial diffusion of FGF9 ^Eks via the intercellular space. This is strongly suggested (FIGS. 6D and 6E).

  Homodimerization has also been reported in the FGF9 / 16/20 subfamily (Mohammadi et al., 2005) and FGF2 (Ornitz et al., 1992; Venkataraman et al., 1999). It is not yet known how much homodimerization affects the activity of other FGFs. The fact that mutations in Fgf9 affected homodimerization, heparin / HS affinity and biological activity is a tool by which drugs that affect FGF homodimerization regulate FGF activity As useful as.

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The present invention provides a new method for determining the risk of developing osteosynthesis. Previously, it was known that mutations in FGFR cause osteosynthesis, but the involvement of FGF mutations, which are its ligands, in osteosynthesis is not known. Therefore, the present invention provides a method for evaluating the onset risk of osteosynthesis from a completely different viewpoint.
The present invention also provides a mutant FGF having a reduced affinity for sulfated glycosaminoglycans. The mutant FGF of the present invention is excellent in diffusibility in the living body and reaches the deep part of the affected area without being trapped by the sulfated glycosaminoglycan in the vicinity of the administration site. Useful as a formulation.

Missense mutation in the Fgf9 gene of Eks mice. (A) Nucleic acid sequence of Fgf9 cDNA from + / + and Eks / Eks mice. The Eks mutant has an A to C substitution at position 428, resulting in a Thr substitution of Asn143. The Eks missense mutation is indicated with an arrowhead and the corresponding amino acid is indicated in purple. (B) Genotyping of Eks mutation. The 189 bp genomic PCR fragment containing the nucleic acid substitution of the Fgf9 gene in the Eks mutant detected in (A) is not digested by BsrI restriction enzyme, but from + / +, C57BL / 6, DBA / 2J and JF1 / Ms mice. The PCR fragment was digested. (C) Synergistic enhancement of lung phenotype in Eks mice by introduction of Fgf9 null allele. The body weight ratio of the E17.5 fetal lung was compared between each genotype. Data are expressed as mean ± standard error of at least 5 embryos. Lung hypoplasia seen in Fgf9 ^+/− and Fgf9 ^{Eks / +} embryos is significantly worse in Eks / − (asterisk) (p <0.001). (D) Change of the state of elbow osteosynthesis in Eks mice by introduction of Fgf9 null allele. Forelimbs at E17.5 of Fgf9 ^{+ / +} , Fgf9 ^{Eks / +} , Fgf9 ^{Eks / −} and Fgf9 ^{Eks / Eks} mice were stained with Alcian Blue and Alizarin Red. The degree of elbow osteosynthesis in Fgf9 ^{Eks / −} mice was between Fgf9 ^{Eks / +} and Fgf9 ^{Eks / Eks} . Ectopic chondrocyte differentiation in the elbow joint planned area of Fgf9 ^{Eks / Eks} mice and abnormal osteoblast localization in the cranial suture. (A) Analysis of joint development in limb buds. Expression of Gdf5 and Col2al genes in the forelimbs of Fgf9 ^{+ / +} and Fgf9 ^{Eks / Eks} fetuses at E10.5 and E11.5 were detected by in situ hybridization. The arrow in g shows the gap | interval of Col2al expression in an elbow joint plan area. Bar, 100 μm. (B) Analysis of cranial suture generation. Sections of cranial sutures of Fgf9 ^{+ / +} and Fgf9 ^{Eks / Eks} fetuses at E15.5 and E16.5 were stained with hematoxylin and eosin (HE) (a, b, g and h), using the indicated probes We examined by in situ hybridization (cf and il). fb, frontal bone; pb, parietal bone. Bar, 100 μm. FGF9 ^Eks are inhibited from dimer formation. The homodimerization of FGF9 ^WT and FGF9 ^Eks was examined by analytical ultracentrifugation. (A and B) Sedimentation equilibrium analysis. The absorbance distribution in the sedimentation equilibrium state at 16000 rpm and 20 ° C. when 0.4 mg / ml FGF9 ^WT (A) and FGF9 ^Eks (B) are used is shown. The solid line is a best-fit plot obtained by global analysis using a non-interaction separation model that assumes a single species. The sedimentation equilibrium data were analyzed with Beckman XL-A / XL-I analysis software. The upper panel is the result of the residual analysis. The average molecular weights of FGF9 ^WT and FGF9 ^Eks were estimated to be 39,264 and 32,929 Da, respectively. Coupling constants of FGF9 ^WT and ^{FGF9 Eks,} respectively, was determined to 10.4MyuM ^-1 and 0.119μM ^-1. (C and D) Sedimentation rate analysis of FGF9 ^WT and FGF9 ^Eks . Precipitation coefficient distribution of FGF9 ^WT (C) and FGF9 ^Eks (D) at ^0.2 , 0.3 and 0.4 mg / ml. FGF9 ^WT shows a unimodal peak at 3.0S. FGF9 ^Eks shows a bimodal peak (2.2S and 3.1S). Reduced heparin affinity of FGF9 ^Eks . The heparin affinity of FGF9 ^WT and FGF9 ^Eks was examined using heparin affinity chromatography (A) and surface plasmon resonance analysis (B and C). (A) FGF9 ^WT and FGF9 ^Eks were loaded onto a heparin-conjugated agarose column and eluted with a linear gradient of NaCl from 120 mM to 2.0 M (black line). The elution of FGF9 ^WT (blue line) and FGF9 ^Eks (red line) was measured by absorbance at 280 nm. (B and C) ^Sensorgram of the interaction of FGF9 ^WT (B) and FGF9 ^Eks (C) with solid phased heparin. The FGF9 ^WT or FGF9 ^Eks concentrations are indicated as follows: 62.5 nM is red, 31.3 nM is brown, 15.6 nM is blue, 7.8 nM is green, 3.9 nM is purple. The biosensor chip reaction is represented by time on the X-axis and ΔRU on the Y-axis. The measurement was performed at room temperature. The homodimerization of FGF9 controls its heparin affinity. The final structure of MD simulation. Heparin and protein residues that form important hydrogen bonds are shown in spheres and bars and space-filling mode. The name of each residue is indicated by a one-letter amino acid code, residue number, and chain code. (A and B) Final structure of MD simulation for dimer FGF9 ^WT (A) and dimer FGF9 ^Eks (B). N143T substitution of FGF9 results in loss of intermonomer hydrogen bonding. (CF) About Dimeric FGF9 ^WT -Heparin (C), ^Dimeric FGF9 ^Eks -Heparin (D), Monomeric FGF9 ^WT -Heparin (E) and Monomeric FGF9 ^Eks -Heparin (F) The final structure of MD simulation. The calculated binding free energy for each complex is shown below the structure. Data are shown as mean values ± standard deviation of energy from each of the 200 MD snapshots. There is a high possibility that joint fusion and early fusion of the skull suture are caused by ectopic signaling of FGF9 ^Eks derived from the high diffusivity of FGF9 ^{Eks in} the tissue. (A) Joint fusion by ectopic expression of Fgf9 ^WT and Fgf9 ^Eks in chicken hindlimb buds. HH stage 17 chickens were infected with RCAS-Fgf9 ^WT , RCAS-Fgf9 ^Eks or empty RCAS virus to induce the expression of Fgf9 ^WT or Fgf9 ^Eks . Two days after infection, hind limbs were examined by in situ hybridization with Fgf9 probe (a and b) and 5 days after infection by staining with alcian blue (ce). Arrows in ce indicate knee joint positions. Ectopic expression of Fgf9 ^WT and Fgf9 ^Eks resulted in knee fusion (cd). Ectopic expression of Fgf9 ^WT resulted in severe hind limb joint fusion (c) and ectopic expression of Fgf9 ^Eks did not result in skeletal atrophy (d). (B) Diffusion ability measurement of FGF9 ^WT and FGF9 ^Eks in the skull primordium. AffiGel-Blue beads containing FGF9 protein were transplanted into the E15 mouse skull, and after 24 hours, Spp1 mRNA was detected by in situ hybridization at E16, and the diffusibility of exogenous FGF9 ^WT and FGF9 ^Eks in the skull primordia was evaluated. . (Ad) Whole mount in situ hybridization for Spp1. The side transplanted with FGF9 ^WT (a) and FGF9 ^Eks (c) beads showed a strong localized signal, indicating the diffusion site of exogenous FGF9 protein. The non-transplanted control sides (b and d) detected only weak signals representing endogenous expression in the frontal and parietal bone primordia. (E) Diffusivity of FGF9 ^WT and FGF9 ^Eks . The diffusion area (%) in the frontal bone primordium and parietal bone primordium was estimated from the area ratio of the strong signal to the total signal. Data are shown as the mean ± standard error of 6 operations (p <0.005). FGF9 ^Eks (red bar) shows higher diffusivity than FGF9 ^WT (blue bar). (C) Examination of diffusivity of FGF9 ^WT and FGF9 ^Eks in E10.5 forelimb buds by implantation of AffiGel-Blue beads containing FGF9 protein. The diffusion of exogenous FGF9 ^WT (a) and FGF9 ^Eks (b) 2 hours after transplantation was detected with FGF9 antibody. (C) Comparison of diffusion areas of FGF9 ^WT and FGF9 ^Eks . Data are expressed as the mean of 5 operations (FGF9 ^WT = 100%) ± standard error. It was statistically significant (p <0.05) that the diffusivity of FGF9 ^Eks (red bar) was higher than that of FGF9 ^WT (blue bar). (D) Onset mechanism of elbow joint fusion in Fgf9 ^{Eks / Eks} mice. In Fgf9 ^{Eks / Eks} mice, ectopic FGF9 signaling by overdiffusion of FGF9 ^Eks in the elbow joint planned area can induce joint fusion. (E) The onset mechanism of skull suture early fusion in Fgf9 ^{Eks / Eks} mice. In Fgf9 ^{Eks / Eks} mice, ectopic FGF9 signaling by overdiffusion of FGF9 ^Eks in the cranial suture can induce early suture fusion. Lung hypoplasia in Eks homozygous mice. (AB) + / + (A) and Eks / Eks (B) mouse lungs at birth. Structure-based sequence alignment of human FGF. The amino acid sequences around the N143T mutation in FGF9Eks and the sequences of the corresponding domains of other human FGF family proteins were aligned based on sequence identity. The amino acid of each FGF protein at a position parallel to Asn143 in FGF9 is indicated by a red box. Purification of FGF9 ^WT and FGF9 ^Eks proteins. (A) Overview of methods used for purification of FGF9 ^WT and FGF9 ^Eks proteins. (B) SDS / PAGE of purified FGF9 ^WT and FGF9 ^Eks . Both FGF9 ^WT and FGF9 ^Eks were detected at a molecular weight of about 20 kDa. On gel filtration chromatography, the molecular weight of FGF9 ^Eks is smaller than the molecular weight of FGF9 ^WT . Samples were applied separately to a Superdex 75 10/300 GL column. The eluted FGF9 ^WT (blue line) and FGF9 ^Eks (red line) were detected by absorbance at 280 nm. The arrow indicates the elution position of the molecular weight marker: 67 kDa, albumin; 43 kDa, ovalbumin; 25 kDa chymotrypsinogen; 13.7 kDa, ribonuclease A. Partial change in signaling ability of FGF9 ^Eks via FGFR. (AG) FGF9 concentration dose-dependent mitogenic activity of FGF9 ^WT and FGF9 ^Eks . BaF3 cells expressing FGFR1b (A), FGFR1c (B), FGFR2b (C), FGFR2c (D), FGFR3b (E), FGFR3c (F) or FGFR4 (G) in the presence of 1 μg / ml heparin And treated with various concentrations of FGF9 ^WT (blue circles) and FGF9 ^Eks (red triangles). Cell proliferation was measured by [ ³ H] thymidine incorporation after 36 hours of culture. Heparin concentration-dependent mitogenic activity of FGF9 ^WT and FGF9 ^Eks . BaF3 cells expressing FGFR1b (H), FGFR1c (I), FGFR2b (J), FGFR2c (K), FGFR3b (L), FGFR3c (M) or FGFR4 (N), 0.2 nM FGF9 ^WT or Treated with various concentrations of heparin in the presence of FGF9 ^Eks . Cell proliferation was determined as described above. The data is the mean value of the triplicate measurement results ± standard error. These data are representative of at least two independent trials. Fgf9 is expressed in myoblasts, while Fgfrs is expressed in mesenchymal cells in the planned elbow joint area. Expression of Fgf9, Fgfr1, Fgfr2, and Fgfr3 in forelimb buds of Fgf9 ^{+ / +} and Fgf9 ^{Eks / Eks} fetuses at both E10.5 and E11.5 was detected by in situ hybridization. Bar, 100 μm. Distal joint fusion of chicken hind limbs after ectopic expression of Fgf9 ^WT and Fgf9 ^Eks . Chicken hind limbs after infection with RCAS-Fgf9 ^WT (A), RCAS-Fgf9 ^Eks (B), or empty RCAS virus (C) were stained with Alcian blue. The arrows in AC indicate the distal joint. FGF9 ^WT and FGF9 ^Eks capacity of AffiGel-Blue beads. FGF9 ^WT or FGF9 ^Eks retention amount of AffiGel-Blue beads was measured. The beads were soaked in FGF9 ^WT or FGF9 ^Eks solution (500 μg / ml) followed by detection of FGF9 by SDS-PAGE and Western blotting analysis. Lane 1-6 Western blotting of one bead immersed in FGF9 ^WT or FGF9 ^Eks solution (500 μg / ml) per lane. Lanes 7, 8, recombinant FGF9 ^WT and FGF9 ^Eks (100 ng). The amount of FGF9 ^WT and FGF9 ^Eks contained in one AffiGel-Blue is approximately equal.

Claims (14)

  A mutant FGF polypeptide in which Asn contributing to intermonomer hydrogen bonding in an FGF homodimer is replaced with Thr.   The polypeptide of claim 1, wherein the FGF is FGF9.   The polypeptide of claim 2, wherein Asn is Asn143.   The polypeptide according to claim 3, comprising the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 12, or 14.   A polynucleotide comprising a nucleotide sequence encoding the polypeptide according to claim 1.   The polynucleotide according to claim 5, comprising the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 11, or 13.   A partial sequence of the nucleotide sequence represented by SEQ ID NO: 1 or 3 consisting of a continuous nucleotide sequence of 15 or more bases containing cytosine of base number 428 of the nucleotide sequence represented by SEQ ID NO: 1 or 3 or a complementary sequence thereof Polynucleotide.   A vector comprising the polynucleotide according to claim 5.   A transformant into which the vector according to claim 8 has been introduced.   A medicament comprising the polypeptide according to claim 1.   A fracture therapeutic agent comprising the polypeptide according to claim 2.   A method for determining the risk of developing bone fusion, comprising detecting the presence or absence of a substitution of Asn143 in a FGF9 polypeptide with Thr or a mutation in a codon encoding Asn143 in a Fgf9 gene to a codon encoding Thr.   A diagnostic agent for risk of developing bone fusion, comprising a reagent for detecting the substitution of Asn143 in Thr of FGF9 polypeptide or the mutation of a codon encoding Asn143 in the Fgf9 gene to a codon encoding Thr.   Testing whether or not the test substance promotes or inhibits homodimerization of FGF, and converts the test substance that promotes or inhibits homodimerization of FGF to sulfated glycosaminoglycan of FGF A method for screening a substance capable of regulating the affinity of FGF for sulfated glycosaminoglycan, comprising obtaining the substance as a substance capable of regulating affinity.