# Patent

## Title
Chimeric fibroblast growth factor 23 proteins and methods of use

## Abstract

The present invention relates to a chimeric protein that includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine fibroblast growth factor (“FGF”) and the C-terminus includes a C-terminal portion of an FGF23 molecule. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. The present invention also relates to pharmaceutical compositions including chimeric proteins according to the present invention, methods for treating a subject suffering from a disorder, and methods of screening for compounds with enhanced binding affinity for the αKlotho-FGF receptor complex involving the use of chimeric proteins of the present invention.

## Background
This application claims priority benefit of U.S. Provisional Patent Application No. 61/656,887, filed Jun. 7, 2012, and U.S. Provisional Patent Application No. 61/664,097, filed Jun. 25, 2012, each of which is hereby incorporated by reference in its entirety.


This invention was made with government support under grant numbers DE13686, DK077276, AG019712, DK091392, and DK067158 awarded by the U.S. National Institutes of Health. The government has certain rights in this invention.


FIELD OF THE INVENTION
The present invention relates to chimeric fibroblast growth factor (“FGF”) proteins and uses thereof.
BACKGROUND OF THE INVENTION
Fibroblast growth factor (FGF) 23, is an endocrine regulator of phosphate homeostasis and vitamin D metabolism, and was originally identified as the mutated gene in patients with the phosphate wasting disorder “autosomal dominant hypophosphatemic rickets” (ADHR) (Anonymous, “Autosomal Dominant Hypophosphataemic Rickets is Associated with Mutations in FGF23,” Nat Genet 26(3):345-348 (2000)). FGF23 inhibits reabsorption of phosphate in the renal proximal tubule by decreasing the abundance of the type II sodium-dependent phosphate transporters NaPi-2A and NaPi-2C in the apical brush border membrane (Baum et al., “Effect of Fibroblast Growth Factor-23 on Phosphate Transport in Proximal Tubules,” Kidney Int 68(3):1148-1153 (2005); Perwad et al., “Fibroblast Growth Factor 23 Impairs Phosphorus and Vitamin D Metabolism In Vivo and Suppresses 25-hydroxyvitamin D-1alpha-hydroxylase Expression In Vitro,” Am J Physiol Renal Physiol 293(5):F1577-1583 (2007); Larsson et al., “Transgenic Mice Expressing Fibroblast Growth Factor 23 under the Control of the Alpha1 (I) Collagen Promoter Exhibit Growth Retardation, Osteomalacia, and Disturbed Phosphate Homeostasis,” Endocrinology 145(7):3087-3094 (2004)). The phosphaturic activity of FGF23 is down-regulated by proteolytic cleavage at the 176RXXR179 (SEQ ID NO: 233) motif, where “XX” is defined as “HT”, corresponding to positions 177 and 178, respectively, of the FGF23 amino acid sequence, producing an inactive N-terminal fragment (Y25 to R179) and a C-terminal fragment (S180 to I251) (Goetz et al., “Molecular Insights into the Klotho-dependent, Endocrine Mode of Action of Fibroblast Growth Factor 19 Subfamily Members,” Mol Cell Biol 27(9):3417-3428 (2007)). αKlotho, a protein first described as an aging suppressor (Kuro-o et al., “Mutation of the Mouse Klotho Gene Leads to a Syndrome Resembling Aging,” Nature 390(6655):45-51 (1997)), is required by FGF23 in its target tissue in order to exert its phosphaturic activity (Kurosu et al., “Regulation of Fibroblast Growth Factor-23 Signaling by Klotho,” J Biol Chem 281(10):6120-6123 (2006); Urakawa et al., “Klotho Converts Canonical FGF Receptor into a Specific Receptor for FGF23,” Nature 444(7120):770-774 (2006)). αKlotho constitutively binds the cognate FGFRs of FGF23, and the binary FGFR-αKlotho complexes exhibit enhanced binding affinity for FGF23 ((Kurosu et al., “Regulation of Fibroblast Growth Factor-23 Signaling by Klotho,” J Biol Chem 281(10):6120-6123 (2006); Urakawa et al., “Klotho Converts Canonical FGF Receptor into a Specific Receptor for FGF23,” Nature 444(7120):770-774 (2006)). In co-immunoprecipitation studies, it was demonstrated that the mature, full-length form of FGF23 (Y25 to I251) but not the inactive N-terminal fragment of proteolytic cleavage (Y25 to R179) binds to binary FGFR-αKlotho complexes (Goetz et al., “Molecular Insights into the Klotho-dependent, Endocrine Mode of Action of Fibroblast Growth Factor 19 Subfamily Members,” Mol Cell Biol 27(9):3417-3428 (2007)).
It was further shown that the mature, full-length form of FGF23 (Y25 to I251) forms a stable ternary complex with the ectodomain of αKlotho and the ligand-binding domain of FGFR1c in solution (Goetz et al., “Isolated C-terminal Tail of FGF23 Alleviates Hypophosphatemia by Inhibiting FGF23-FGFR-Klotho Complex Formation,” Proc Natl Acad Sci USA 107:407-412 (2010)). The ligand interacts with a de novo binding site generated at the composite receptor-coreceptor interface in the binary αKlotho-FGFR complex (Goetz et al., “Isolated C-terminal Tail of FGF23 Alleviates Hypophosphatemia by Inhibiting FGF23-FGFR-Klotho Complex Formation,” Proc Natl Acad Sci USA 107:407-412 (2010)). The region on FGF23 that binds to this de novo site was mapped to the 72 amino acid long C-terminal tail, which follows the β-trefoil core domain (Goetz et al., “Isolated C-terminal Tail of FGF23 Alleviates Hypophosphatemia by Inhibiting FGF23-FGFR-Klotho Complex Formation,” Proc Natl Acad Sci USA 107:407-412 (2010)). Thus, the N-terminal fragment of proteolytic cleavage (Y25 to R179) is metabolically inactive because it lacks the binding site for the αKlotho-FGFR complex. The C-terminal proteolytic fragment (S180 to I251), however, can compete with full-length FGF23 for binding to the αKlotho-FGFR complex to antagonize the metabolic activity of FGF23, because this fragment contains the binding site for the αKlotho-FGFR complex (Goetz et al., “Isolated C-terminal Tail of FGF23 Alleviates Hypophosphatemia by Inhibiting FGF23-FGFR-Klotho Complex Formation,” Proc Natl Acad Sci USA 107:407-412 (2010)). These findings suggest a dual mechanism by which proteolytic cleavage at the RXXR motif inactivates FGF23: the cleavage removes the binding site for the αKlotho-FGFR complex from FGF23 and concomitantly generates an endogenous inhibitor of FGF23 Inhibition of this proteolytic cleavage by missense mutations at the RXXR motif in FGF23 leads to accumulation of full-length, bioactive FGF23, causing renal phosphate wasting disease in humans (Shimada et al., “Mutant FGF-23 Responsible for Autosomal Dominant Hypophosphatemic Rickets is Resistant to Proteolytic Cleavage and Causes Hypophosphatemia in vivo,” Endocrinology 143:3179-3182 (2002); White et al., “Autosomal-dominant Hypophosphatemic Rickets (ADHR) Mutations Stabilize FGF-23,” Kidney Int 60:2079-2086 (2001); White et al., “Autosomal Dominant Hypophosphataemic Rickets is Associated with Mutations in FGF23,” Nature Genet 26:345-348 (2000)).
Conversely, enhanced FGF23 cleavage due to impaired O-glycosylation of FGF23 leads to a deficit in full-length FGF23, which manifests as hyperphosphatemia and soft tissue calcification in humans (Frishberg Y et al., “Hyperostosis-hyperphosphatemia Syndrome: a Congenital Disorder of O-glycosylation Associated with Augmented Processing of Fibroblast Growth Factor 23,” J Bone Miner Res 22:235-242 (2007); Kato et al., “Polypeptide GalNAc-transferase T3 and Familial Tumoral Calcinosis. Secretion of Fibroblast Growth Factor 23 Requires β-glycosylation,” J Biol Chem 281:18370-18377 (2006)). Familial tumoral calcinosis is an autosomal recessive metabolic disorder associated with hyperphosphatemia and soft tissue calcification. Missense mutations in either the UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylglactosaminyltransferase 3 (GALNT3) gene (Garringer et al., “Two Novel GALNT3 Mutations in Familial Tumoral Calcinosis,” Am Med Genet A 143A:2390-2396 (2007)) or the FGF23 gene (Garringer et al., “Molecular Genetic and Biochemical Analyses of FGF23 Mutations in Familial Tumoral Calcinosis,” Am Physiol Endocrinol Metab 295:E929-E937 (2008); Araya et al., “A Novel Mutation in Fibroblast Growth Factor 23 Gene as a Cause of Tumoral Calcinosis,” J Clin Endocrinol Metab 90:5523-5527 (2005)) have been associated with familial tumoral calcinosis. There is a great need for suitable treatments for such patients.
The present invention is directed to overcoming these and other deficiencies in the art.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to a chimeric protein. The chimeric protein includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine fibroblast growth factor (“FGF”) and the C-terminus includes a C-terminal portion of an FGF23 molecule. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification.
Another aspect of the present invention relates to a method for treating a subject suffering from a disorder. This method involves selecting a subject suffering from the disorder. The method also involves providing a chimeric FGF protein, where the chimeric FGF protein includes an N-terminus coupled to a C-terminus. The N-terminus includes a portion of a paracrine FGF and the C-terminus includes a C-terminal portion of FGF23. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves administering a therapeutically effective amount of the chimeric FGF protein to the selected subject under conditions effective to treat the disorder.
Another aspect of the present invention relates to a method of making a chimeric FGF protein possessing enhanced endocrine activity. This method involves introducing one or more modifications to an FGF protein, where the modification decreases the affinity of the FGF protein for heparin and/or heparan sulfate and coupling a C-terminal portion of FGF23 that includes an α-Klotho-FGFR complex binding domain to the modified FGF protein's C-terminus.
Yet another aspect of the present invention relates to a method of facilitating fibroblast growth factor receptor (“FGFR”)-α-Klotho co-receptor complex formation. This method involves providing a cell that includes a α-Klotho co-receptor and an FGFR and providing a chimeric FGF protein. The chimeric FGF protein includes a C-terminal portion of FGF23 and a portion of a paracrine FGF, where the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves contacting the cell and the chimeric FGF protein under conditions effective to cause FGFR-αKlotho co-receptor complex formation.
Yet a further aspect of the present invention relates to a method of screening for agents capable of facilitating fibroblast growth factor receptor (“FGFR”)-αKlotho co-receptor complex formation in the treatment of a disorder. This method involves providing a chimeric FGF that includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine FGF and the C-terminus includes a C-terminal portion of FGF23. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves providing a binary αKlotho-FGFR complex and providing one or more candidate agents. This method further involves combining the chimeric FGF, the binary αKlotho-FGFR complex, and the one or more candidate agents under conditions permitting the formation of a ternary complex between the chimeric FGF and the binary αKlotho-FGFR complex in the absence of the one or more candidate agents. This method also involves identifying the one or more candidate agents that decrease ternary complex formation between the chimeric FGF and the binary αKlotho-FGFR compared to the ternary complex formation in the absence of the one or more candidate agents as suitable for treating the disorder.
Yet another aspect of the present invention relates to a modified FGF23 protein. The modified FGF23 protein includes an FGF23 protein that includes a modification to decrease binding affinity for heparin and/or heparan sulfate compared to an FGF23 protein without the modification.
Another aspect of the present invention relates to a method for treating a subject suffering from a disorder. This method involves selecting a subject suffering from the disorder and administering to the selected subject a therapeutically effective amount of a modified FGF23 protein that includes a modification to decrease binding affinity for heparin and/or heparan sulfate compared to an FGF23 protein without the modification.
Fibroblast growth factors (FGFs) 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. To decipher the role of HS in endocrine FGF signaling, we generated FGF19 and FGF23 mutant ligands devoid of HS binding and compared their signaling capacity with that of wild-type ligands. The data presented herein show that the mutated ligands retain full metabolic activity demonstrating that HS does not participate in the formation of the endocrine FGF signaling complex. Here it is shown that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. A paracrine FGF is converted into an endocrine ligand by diminishing heparan sulfate binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor binding site in order to home the ligand into the target tissue. The ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1D are schematic diagrams showing side-by-side comparison of the HS-binding site of FGF2, FGF19, and FGF23, and working model of the endocrine FGF signaling complex. FIG. 1A shows interactions of FGF2 (schematic representation) with a heparin hexasaccharide (shown as sticks) as observed in the crystal structure of the 2:2 FGF2-FGFR1c dimer (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)). The heparin hexasaccharide consists of three disaccharide units of 1→4 linked N-sulfated-6-O-sulfated D-glucosamine and 2-O-sulfated L-iduronic acid. Note that the heparin hexasaccharide interacts with both side chain and backbone atoms of residues in the HS-binding site of FGF2. Dashed lines denote hydrogen bonds. K128, R129, and K134, which make the majority of hydrogen bonds with the heparin hexasaccharide, are boxed. The β-strand nomenclature follows the original FGF1 and FGF2 crystal structures (Ago et al., J. Biochem. 110:360-363 (1991); Eriksson et al., Proc. Nat'l. Acad. Sci. U.S.A. 88:3441-3445 (1991); Zhang et al., Proc. Nat'l. Acad. Sci. U.S.A. 88:3446-3450 (1991); Zhu et al., Science 251:90-93 (1991), which are hereby incorporated by reference in their entirety). Please note that compared to the prototypical β-trefoil fold seen in soybean trypsin inhibitor (PDB ID: 1TIE; (Onesti et al., J. Mol. Biol. 217:153-176 (1991), which is hereby incorporated by reference in its entirety)) and interleukin 1β (PDB ID: 1I1B; (Finzel et al., J. Mol. Biol. 209:779-791 (1989), which is hereby incorporated by reference in its entirety)), the β10-β11 strand pairing in FGF2 and other paracrine FGFs is less well defined. FIGS. 1B and 1C show cartoon representation of the crystal structures of FGF19 (PDB ID: 2P23; (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety)) (FIG. 1B) and FGF23 (PDB ID: 2P39; (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety)) (FIG. 1C) shown in the same orientation as the FGF2 structure in FIG. 1A. Side chains of residues that map to the corresponding HS-binding sites of these ligands are shown as sticks. Residues selected for mutagenesis to knock out residual HS binding in FGF19 and FGF23 are boxed. NT and CT indicate N- and C-termini of the FGFs. FIG. 1D is a schematic of two working models for the endocrine FGF-FGFR-Klotho coreceptor signal transduction unit. A recent study on the ternary complex formation between FGF21, FGFR1c, and βKlotho supports the 1:2:1 model rather than the 2:2:2 model (Ming et al., J. Biol. Chem. 287:19997-20006 (2012), which is hereby incorporated by reference in its entirety). For comparison, a schematic of the paracrine FGF-FGFR-HS signaling unit is shown, which was made based on the crystal structure of the 2:2:2 FGF2-FGFR1c-HS complex (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)). HS engages both paracrine FGF and receptor to enhance binding of FGF to its primary and secondary receptors thus promoting receptor dimerization. A question mark denotes whether or not HS is also a component of the endocrine FGF signaling complex.
FIG. 2 shows a sequence alignment of the endocrine FGFs, FGF1, and FGF2. The amino acid sequences of the mature human FGF19, FGF21, and FGF23 ligands are aligned. Also included in the alignment are the human sequences of FGF1 and FGF2, prototypical paracrine FGFs, which were used in the experiments described herein, in which FGF1 and FGF2 were converted into endocrine FGF ligands. Residue numbers corresponding to the human sequence of FGF1 (SEQ ID NO: 1) (GenBank Accession No. AAH32697, which is hereby incorporated by reference in its entirety), FGF2 (SEQ ID NO: 121) (GenBank Accession No. EAX05222, which is hereby incorporated by reference in its entirety), FGF19 (SEQ ID NO: 333) (GenBank Accession No. NP_005108, which is hereby incorporated by reference in its entirety), FGF21 (SEQ ID NO: 334) (GenBank Accession No. NP_061986, which is hereby incorporated by reference in its entirety), and FGF23 (SEQ ID NO: 233) (GenBank accession no. AAG09917, which is hereby incorporated by reference in its entirety) are in parenthesis to the left of the alignment. Secondary structure elements are labeled, and residues containing these elements for known secondary structures are boxed. Gaps (dashes) were introduced to optimize the sequence alignment. The β-trefoil core domain for known FGF crystal structures is shaded gray. Blue bars on top of the alignment indicate the location of the HS-binding regions. HS-binding residues selected for mutagenesis are shaded blue.
FIGS. 3A-3G show Surface plasmon resonance (“SPR”) results relating to knockout of residual heparin binding in FGF19 and FGF23 by site-directed mutagenesis. FIG. 3A shows an overlay of SPR sensorgrams illustrating heparin binding of FGF2, FGF19, FGF21, and FGF23 (left panel) and an exploded view of the binding responses for FGF19-, FGF21-, and FGF23-heparin interactions (right panel). Heparin was immobilized on a biosensor chip, and 400 nM of FGF2, FGF19, FGF21, or FGF23 were passed over the chip. Note that FGF19, FGF21, and FGF23 exhibit measurable, residual heparin binding and that differences in heparin binding exist between these three endocrine FGFs. FIGS. 3B-3D show overlays of SPR sensorgrams illustrating binding of FGF19 to heparin (FIG. 3B) and lack of interaction between the FGF19K149A mutant and heparin (FIG. 3C) and between the FGF19K149A, R157A mutant and heparin (FIG. 3D). Heparin was immobilized on a biosensor chip, and increasing concentrations of FGF19 were passed over the chip. Thereafter, FGF19K149A or FGF19K149A, R157A was injected over the heparin chip at the highest concentration tested for the wild-type ligand. FIGS. 3E-3G show overlays of SPR sensorgrams illustrating binding of FGF23 to heparin (FIG. 3E), poor interaction between the FGF23R48A, N49A mutant and heparin (FIG. 3F), and lack of interaction between the FGF23R140A, R143A mutant and heparin (FIG. 3G). Heparin was immobilized on a biosensor chip, and increasing concentrations of FGF23 were passed over the chip. FGF23R48A, N49A or FGF23R140A, R143A was then injected over the heparin chip at the highest concentration tested for the wild-type ligand.
FIGS. 4A-4D show results demonstrating that HS is dispensable for the metabolic activity of FGF19 and FGF23. FIG. 4A shows results of an immunoblot analysis of phosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) in H4IIE hepatoma cells following stimulation with the FGF19K149A mutant, the FGF19K149A, R157A mutant, or wild-type FGF19. Numbers above the lanes give the amounts of protein added in ng ml−1. Total 44/42 MAPK protein expression was used as a loading control. FIG. 4B shows results of an immunoblot analysis of phosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) in a HEK293-αKlotho cell line following stimulation with the FGF23R48A, N49A mutant, the FGF23R140A, R143A mutant, or wild-type FGF23. Numbers above the lanes give the amounts of protein added in ng ml−1. Total 44/42 MAPK and αKlotho protein expression were used as loading controls. FIG. 4C shows graphical results of a quantitative analysis of CYP7A1 and CYP8B1 mRNA expression in liver tissue from mice treated with FGF19K149A, FGF19K149A, R157A, FGF19, or vehicle. 1 mg of protein per kg of body weight was given. Data are presented as mean±SEM; ***, P<0.001 by Student's t test. FIG. 4D shows graphical results of analysis of serum phosphate concentrations (serum Pi) in mice before and 8 h after intraperitoneal injection of FGF23R48A, N49A, FGF23R140A, R143A, FGF23, or vehicle. Wild-type mice were given a single dose of protein (0.29 mg kg body weight−1), whereas Fgf23 knockout mice received two doses of 0.71 mg kg body weight−1 each. Data are presented as mean±SEM; *, P<0.05, and **, P<0.01 by ANOVA.
FIGS. 5A-5G show design and results relating to the conversion of FGF2 into an endocrine ligand. FIG. 5A is a schematic of human FGF2, FGF19, FGF21, FGF23, and engineered FGF2-FGF19, FGF2-FGF21, and FGF2-FGF23 chimeras. Amino acid boundaries of each ligand and of each component of the chimeras are labeled with residue letter and number. The β-trefoil core domain for the known ligand crystal structures is shaded gray. HS-binding residues mutated in the FGF2 portion of chimeras are labeled with residue letter and number. Also labeled are the arginine residues of the proteolytic cleavage site in the C-terminal region of FGF23 that were mutated to glutamine in both FGF23 and the FGF2-FGF23 chimeras. FIGS. 5B and 5C show overlays of SPR sensorgrams illustrating binding of FGF2WTcore-FGF21C-tail (FIG. 5B) and FGF2ΔHBScore-FGF21C-tail (FIG. 5C) to heparin, and fitted saturation binding curves. Heparin was immobilized on a biosensor chip, and increasing concentrations of FGF2WTcore-FGF21C-tail or FGF2ΔHBScore-FGF21C-tail were passed over the chip. Dissociation constants (KDs) were derived from the saturation binding curves. FIGS. 5D and 5E show overlays of SPR sensorgrams illustrating binding of FGF2WTcore-FGF23C-tail (FIG. 5D) and FGF2ΔHBScore-FGF23C-tail (FIG. 5E) to heparin. Increasing concentrations of FGF2WTcore-FGF23C-tail or FGF2ΔHBScore-FGF23C-tail were passed over a chip containing immobilized heparin. FIGS. 5F and 5G show results of immunoblot analysis for Egr1 expression in HEK293 cells following stimulation with chimeras or native FGFs as denoted. Numbers above the lanes give the amounts of protein added in nanomolar. GAPDH protein expression was used as a loading control.
FIG. 6 is a schematic illustrating the conversion of FGF1 into an endocrine ligand. Shown are schematic drawings of human FGF1, FGF19, FGF21, FGF23, and exemplary FGF1-FGF19, FGF1-FGF21, and FGF1-FGF23 chimeras according to the present invention. Amino acid boundaries of each ligand and of each component of the chimeras are labeled with residue letter and number. The β-trefoil core domain for the known ligand crystal structures is shaded gray. HS-binding residues mutated in the FGF1 portion of chimeras are labeled with residue letter and number. Also labeled are the arginine residues of the proteolytic cleavage site in the C-terminal region of FGF23 that were mutated to glutamine in both FGF23 and the FGF1-FGF23 chimeras.
FIGS. 7A-7G show results demonstrating that the FGF2ΔHBScore-FGF23C-tail chimera exhibits FGF23-like activity. FIGS. 7A and 7B show overlays of SPR sensorgrams illustrating inhibition by FGF2ΔHBScore-FGF23C-tail (FIG. 7A) or FGF23 (FIG. 7B) of αKlotho-FGFR1c binding to FGF23 immobilized on a biosensor chip. Increasing concentrations of FGF2ΔHBScore-FGF23C-tail or FGF23 were mixed with a fixed concentration of αKlotho-FGFR1c complex, and the mixtures were passed over a FGF23 chip. FIG. 7C shows an overlay of SPR sensorgrams illustrating failure of FGF2 to inhibit αKlotho-FGFR1c binding to FGF23. FGF2 and αKlotho-FGFR1c complex were mixed at a molar ratio of 15:1, and the mixture was passed over a biosensor chip containing immobilized FGF23. FIGS. 7D and 7E show overlays of SPR sensorgrams illustrating no inhibition by FGF2ΔHBScore-FGF23C-tail (FIG. 7D) or FGF23 (FIG. 7E) of βKlotho-FGFR1c binding to FGF21. FGF2ΔHBScore-FGF23C-tail or FGF23 were mixed with βKlotho-FGFR1c complex at a molar ratio of 10:1, and the mixtures were passed over a biosensor chip containing immobilized FGF21. FIG. 7F shows analysis of serum phosphate concentrations (serum Pi) in mice before and 8 h after intraperitoneal injection of FGF2ΔHBScore-FGF23C-tail, FGF2WTcore-FGF23C-tail, FGF23, or vehicle. Wild-type mice and αKlotho knockout mice were given 0.21 mg and 0.51 mg of protein, respectively, per kg of body weight. Data are presented as mean±SEM; **, P<0.01; ***, P<0.001 by ANOVA. FIG. 7G shows quantitative analysis of CYP27B1 mRNA expression in renal tissue from mice injected with FGF2ΔHBScore-FGF23C-tail, FGF2WTcore-FGF23C-tail, FGF23, or vehicle. 0.21 mg of protein per kg of body weight were injected. Data are presented as mean±SEM; ***, P<0.001 by ANOVA.
FIGS. 8A-8G show results demonstrating that the FGF2ΔHBScore-FGF21C-tail chimera exhibits FGF21-like activity. FIGS. 8A-8B show overlays of SPR sensorgrams illustrating inhibition by FGF2ΔHBScore-FGF21C-tail (FIG. 8A) or FGF21 (FIG. 8B) of βKlotho-FGFR1c binding to FGF21 immobilized on a biosensor chip. Increasing concentrations of FGF2ΔHBScore-FGF21C-tail or FGF21 were mixed with a fixed concentration of βKlotho-FGFR1c complex, and the mixtures were passed over a FGF21 chip. FIG. 8C shows an overlay of SPR sensorgrams illustrating failure of FGF2 to inhibit βKlotho-FGFR1c binding to FGF21. FGF2 and βKlotho-FGFR1c complex were mixed at a molar ratio of 15:1, and the mixture was passed over a biosensor chip containing immobilized FGF21. FIGS. 8D-8E show overlays of SPR sensorgrams illustrating no inhibition by FGF2ΔHBScore-FGF21C-tail (FIG. 8D) or FGF21 (FIG. 8E) of αKlotho-FGFR1c binding to FGF23. FGF2ΔHBScore-FGF21C-tail or FGF21 were mixed with αKlotho-FGFR1c complex at a molar ratio of 10:1, and the mixtures were passed over a biosensor chip containing immobilized FGF23. FIG. 8F shows results of immunoblot analysis for Egr1 expression in HEK293-βKlotho cells stimulated with FGF2ΔHBScore-FGF21C-tail or FGF21. Numbers above the lanes give the amounts of protein added in ng ml−1. GAPDH protein expression was used as a loading control. Note that the FGF2ΔHBScore-FGF21C-tail chimera is more potent than native FGF21 at inducing Egr1 expression suggesting that the chimera has agonistic property. This is expected since the core domain of FGF2 has inherently greater binding affinity for FGFR than the core domain of FGF21 (see FIGS. 10A and 10C). FIG. 8G shows graphical results of analysis of blood glucose concentrations in mice before and at the indicated time points after intraperitoneal injection of insulin alone, insulin plus FGF2ΔHBScore-FGF21C-tail chimera, insulin plus FGF21, or vehicle alone. 0.5 units of insulin per kg of body weight and 0.3 mg of FGF21 ligand per kg of body weight were injected. Blood glucose concentrations are expressed as percent of pre-injection values. Data are presented as mean±SEM.
FIGS. 9A-9C show the glucose-lowering effects in ob/ob mice of FGF1 variants according to the present invention. FIG. 9A shows graphical results of analysis of blood glucose concentrations in ob/ob mice before and at the indicated time points after subcutaneous injection of FGF1 or FGF21. FIG. 9B shows graphical results of analysis of blood glucose concentrations in ob/ob mice before and at the indicated time points after subcutaneous injection of FGF1, FGF1ΔNT, or FGF1ΔHBS. FIG. 9C shows graphical results of analysis of blood glucose concentrations in ob/ob mice before and at the indicated time points after subcutaneous injection of FGF1 or FGF1ΔHBScore-FGF21C-tail chimera. For the experiments shown in FIGS. 9A-9C, ob/ob mice were injected with a bolus of 0.5 mg of FGF protein per kg of body weight. Data are presented as mean±SD.
FIGS. 10A-10F show results demonstrating that endocrine FGFs have low binding affinity for FGFR1c compared to FGF2. FIGS. 10A-10D show overlays of SPR sensorgrams illustrating binding of FGFR1c to FGF2 (FIG. 10A), FGF19 (FIG. 10B), FGF21 (FIG. 10C), and FGF23 (FIG. 10D), and fitted saturation binding curves. Increasing concentrations of FGFR1c ligand-binding domain were passed over a biosensor chip containing immobilized FGF2, FGF19, FGF21, or FGF23. FIG. 10E shows an overlay of SPR sensorgrams illustrating binding of αKlotho-FGFR1c complex to FGF23. Increasing concentrations of αKlotho-FGFR1c complex were passed over a biosensor chip containing immobilized FGF23. FIG. 10F shows an overlay of SPR sensorgrams showing lack of interaction between the C-terminal tail peptide of FGF23 and FGFR1c. FGF23C-tail was immobilized on a biosensor chip and increasing concentrations of FGFR1c ligand-binding domain were passed over the chip. Dissociation constants (KDs) given in FIGS. 10A-10E were derived from the saturation binding curves.
FIG. 11 shows an alignment of the C-terminal tail sequences of human FGF19 (GenBank Accession No. NP13 005108, which is hereby incorporated by reference in its entirety), FGF21 (GenBank Accession No. NP_061986, which is hereby incorporated by reference in its entirety), and FGF23 (GenBank accession no. AAG09917, which is hereby incorporated by reference in its entirety). Residue numbers are in parenthesis to the left of the alignment. Gaps (dashes) were introduced to optimize the alignment. Residues that are identical between FGF19 and FGF21 are shaded gray. Note that 40% of these residues map to the most C-terminal sequence.



DETAILED DESCRIPTION OF THE INVENTION
One aspect of the present invention relates to a chimeric protein. The chimeric protein includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine fibroblast growth factor (“FGF”) and the C-terminus includes a C-terminal portion of an FGF23 molecule. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification.
As described by Goetz et al. (Goetz et al., “Molecular Insights into the Klotho-Dependent, Endocrine Mode of Action of Fibroblast Growth Factor 19 Subfamily Members,” Mol Cell Biol 3417-3428 (2007), which is hereby incorporated by reference in its entirety), the mammalian fibroblast growth factor (FGF) family comprises 18 polypeptides (FGF1 to FGF10 and FGF16 to FGF23), which participate in a myriad of biological processes during embryogenesis, including but not limited to gastrulation, body plan formation, somitogenesis, and morphogenesis of essentially every tissue/organ such as limb, lung, brain, and kidney (Bottcher et al., “Fibroblast Growth Factor Signaling During Early Vertebrate Development,” Endocr Rev 26:63-77 (2005), and Thisse et al., “Functions and Regulations of Fibroblast Growth Factor Signaling During Embryonic Development,” Dev Biol 287:390-402 (2005), which are hereby incorporated by reference in their entirety).
FGFs execute their biological actions by binding to, dimerizing, and activating FGFR tyrosine kinases, which are encoded by four distinct genes (Fgfr1 to Fgfr4). Prototypical FGFRs consist of an extracellular domain composed of three immunoglobulin-like domains, a single-pass transmembrane domain, and an intracellular domain responsible for the tyrosine kinase activity (Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev 16:107-137 (2005), which is hereby incorporated by reference in its entirety).
The number of principal FGFRs is increased from four to seven due to a major tissue-specific alternative splicing event in the second half of the immunoglobulin-like domain 3 of FGFR1 to FGFR3, which creates epithelial lineage-specific “b” and mesenchymal lineage-specific “c” isoforms (Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev 16:107-137 (2005) and Ornitz et al., “Fibroblast Growth Factors,” Genome Biol 2(3):reviews3005.1-reviews3005.12 (2001), which are hereby incorporated by reference in their entirety). Generally, the receptor-binding specificity of FGFs is divided along this major alternative splicing of receptors whereby FGFRb-interacting FGFs are produced by epithelial cells and FGFRc-interacting FGFs are produced by mesenchymal cells (Ornitz et al., “Fibroblast Growth Factors,” Genome Biol 2(3):reviews3005.1-reviews3005.12 (2001), which is hereby incorporated by reference in its entirety). These reciprocal expression patterns of FGFs and FGFRs result in the establishment of specific paracrine FGF signaling loops between the epithelium and the mesenchyme, which is essential for proper organogenesis and patterning during embryonic development as well as tissue homeostasis in the adult organism.
Based on sequence homology and phylogenetic and structural considerations, the eighteen mammalian FGFs are grouped into six subfamilies (Itoh et al., “Fibroblast growth factors: from molecular evolution to roles in development, metabolism, and disease,” J Biochem 149:121-130 (2011); Mohammadi et al., “Structural basis for fibroblast growth factor receptor activation,” Cytokine Growth Factor Rev 16:107-137 (2005), which are hereby incorporated by reference in its entirety). The FGF core homology domain (approximately 120 amino acids long) is flanked by N- and C-terminal sequences that are highly variable in both length and primary sequence, particularly among different FGF subfamilies. The core region of FGF19 shares the highest sequence identity with FGF21 (38%) and FGF23 (36%), and therefore, these ligands are considered to form a subfamily.
Based on mode of action, the eighteen mammalian FGFs are grouped into paracrine-acting ligands (five FGF subfamilies) and endocrine-acting ligands (one FGF subfamily) comprising FGF19, FGF21 and FGF23 (Itoh and Ornitz, “Fibroblast Growth Factors: From Molecular Evolution to Roles in Development, Metabolism and Disease,” J. Biochem. 149:121-130 (2011); Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev. 16:107-137 (2005), which are hereby incorporated by reference in their entirety).
Paracrine FGFs direct multiple processes during embryogenesis, including gastrulation, somitogenesis, organogenesis, and tissue patterning (Itoh and Ornitz, “Fibroblast Growth Factors: From Molecular Evolution to Roles in Development, Metabolism and Disease,” J. Biochem. 149:121-130 (2011); Bottcher and Niehrs, “Fibroblast Growth Factor Signaling During Early Vertebrate Development,” Endocr. Rev. 26:63-77 (2005); Thisse et al., “Functions and Regulations of Fibroblast Growth Factor Signaling During Embryonic Development,” Dev. Biol. 287:390-402 (2005), which are hereby incorporated by reference in their entirety), and also regulate tissue homeostasis in the adult (Hart et al., “Attenuation of FGF Signalling in Mouse Beta-cells Leads to Diabetes,” Nature 408:864-868 (2000); Jonker et al., “A PPARγ-FGF1 Axis is Required for Adaptive Adipose Remodelling and Metabolic Homeostasis,” Nature 485:391-394 (2012), which is hereby incorporated by reference in its entirety).
Endocrine FGFs control major metabolic processes such as bile acid homeostasis (Inagaki et al., “Fibroblast Growth Factor 15 Functions as an Enterohepatic Signal to Regulate Bile Acid Homeostasis,” Cell Metab. 2:217-225 (2005), which is hereby incorporated by reference in its entirety), and hepatic glucose and protein metabolism (Kir et al., “FGF19 as a Postprandial, Insulin-Independent Activator of Hepatic Protein and Glycogen Synthesis,” Science 331:1621-1624 (2011); Potthoff et al., “FGF15/19 Regulates Hepatic Glucose Metabolism by Inhibiting the CREB-PGC-1α Pathway,” Cell Metab. 13:729-738 (2011), which are hereby incorporated by reference in their entirety) (FGF19), glucose and lipid metabolism (Badman et al., “Hepatic Fibroblast Growth Factor 21 Is Regulated by PPARα and Is a Key Mediator of Hepatic Lipid Metabolism in Ketotic States,” Cell Metab. 5:426-437 (2007); Inagaki et al., “Endocrine Regulation of the Fasting Response by PPARalpha-mediated Induction of Fibroblast Growth Factor 21,” Cell Metab. 5:415-425 (2007); Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J. Clin. Invest. 115:1627-1635 (2005); Potthoff et al., “FGF21 Induces PGC-1alpha and Regulates Carbohydrate and Fatty Acid Metabolism During the Adaptive Starvation Response,” Proc. Nat'l. Acad. Sci. U.S.A. 106:10853-10858 (2009), which are hereby incorporated by reference in their entirety) (FGF21), and phosphate and vitamin D homeostasis (White et al., “Autosomal Dominant Hypophosphataemic Rickets is Associated with Mutations in FGF23,” Nat. Genet. 26:345-348 (2000); Shimada et al., “Targeted Ablation of Fgf23 Demonstrates an Essential Physiological Role of FGF23 in Phosphate and Vitamin D Metabolism,” J. Clin. Invest. 113:561-568 (2004), which are hereby incorporated by reference in their entirety) (FGF23). Thus, these ligands have attracted much attention as potential drugs for the treatment of various inherited or acquired metabolic disorders (Beenken and Mohammadi, “The FGF Family: Biology, Pathophysiology and Therapy,” Nat. Rev. Drug Discov. 8:235-253 (2009); Beenken and Mohammadi, “The Structural Biology of the FGF19 Subfamily,” in Endocrine FGFs and Klothos (Kuro-o, M. ed.), Landes Bioscience. pp 1-24 (2012), which are hereby incorporated by reference in their entirety).
FGFs share a core homology region of about one hundred and twenty amino acids that fold into a β-trefoil (Ago et al., J. Biochem. 110:360-363 (1991); Eriksson et al., Proc. Nat'l. Acad. Sci. U.S.A. 88:3441-3445 (1991); Zhang et al., Proc. Nat'l. Acad. Sci. U.S.A. 88:3446-3450 (1991); Zhu et al., Science 251:90-93 (1991), which are hereby incorporated by reference in their entirety) consisting of twelve β strands in paracrine FGFs (β1-β12) and eleven β strands in endocrine FGFs (β1-β10 and β12) (Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev. 16:107-137 (2005); Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which are hereby incorporated by reference in their entirety). The conserved core region is flanked by divergent N- and C-termini, which play a critical role in conferring distinct biological activity on FGFs (Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev. 16:107-137 (2005); Olsen et al., Genes Dev. 20:185-198 (2006), which are hereby incorporated by reference in their entirety).
All FGFs interact with pericellular heparan sulfate (HS) glycosaminoglycans albeit with different affinities (Asada et al., Biochim. Biophys. Acta. 1790:40-48 (2009), which is hereby incorporated by reference in its entirety). The HS-binding site of FGFs is comprised of the β1-β2 loop and the region between β10 and β12 strands (Mohammadi et al., “Structural Basis for Fibroblast Growth Factor Receptor Activation,” Cytokine Growth Factor Rev. 16:107-137 (2005), which is hereby incorporated by reference in its entirety). HS interacts with both side chain and main chain atoms of the HS-binding site in paracrine FGFs (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety). The HS-binding site of endocrine FGFs deviates from the common conformation adopted by paracrine FGFs such that interaction of HS with backbone atoms of the HS-binding site is precluded (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety). As a result, compared to paracrine FGFs, endocrine FGFs exhibit poor affinity for HS (Beenken and Mohammadi, “The FGF Family: Biology, Pathophysiology and Therapy,” Nat. Rev. Drug Discov. 8:235-253 (2009); Asada et al., Biochim. Biophys. Acta. 1790:40-48 (2009), which are hereby incorporated by reference in their entirety). The poor HS affinity enables these ligands to diffuse freely away from the site of their secretion and enter the blood circulation to reach their distant target organs (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007); Asada et al., Biochim. Biophys. Acta. 1790:40-48 (2009), which are hereby incorporated by reference in their entirety).
By contrast, owing to their high HS affinity (Asada et al., Biochim. Biophys. Acta. 1790:40-48 (2009), which is hereby incorporated by reference in its entirety), paracrine FGFs are mostly immobilized in the vicinity of the cells secreting these ligands, and hence can only act within the same organ. There is emerging evidence that differences in HS-binding affinity among paracrine FGFs translate into the formation of ligand-specific gradients in the pericellular matrix (Kalinina et al., Mol. Cell Biol. 29:4663-4678 (2009); Makarenkova et al., Sci. Signal 2:ra55 (2009), which are hereby incorporated by reference in their entirety), which contribute to the distinct functions of these ligands (Beenken and Mohammadi, “The FGF Family: Biology, Pathophysiology and Therapy,” Nat. Rev. Drug Discov. 8:235-253 (2009); Itoh and Ornitz, “Fibroblast Growth Factors: From Molecular Evolution to Roles in Development, Metabolism and Disease,” J. Biochem. 149:121-130 (2011), which are hereby incorporated by reference in their entirety).
Besides controlling ligand diffusion in the extracellular space, HS promotes the formation of the 2:2 paracrine FGF-FGFR signal transduction unit (Schlessinger et al., Mol. Cell 6:743-750 (2000); Mohammadi et al., Curr. Opin. Struct. Biol. 15:506-516 (2005), which are hereby incorporated by reference in their entirety). HS engages both ligand and receptor to enhance the binding affinity of FGF for receptor and promote dimerization of ligand-bound receptors. Owing to their poor HS-binding affinity, endocrine FGFs rely on Klotho co-receptors to bind their cognate FGFR (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007); Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Ogawa et al., Proc. Nat'l. Acad. Sci. U.S.A. 104:7432-7437 (2007); Urakawa et al., Nature 444:770-774 (2006), which are hereby incorporated by reference in their entirety). Klotho co-receptors are single-pass transmembrane proteins with an extracellular domain composed of two type I β-glycosidase domains (Ito et al., Mech. Dev. 98:115-119 (2000); Kuro-o et al., Nature 390:45-51 (1997), which are hereby incorporated by reference in their entirety). Klotho co-receptors constitutively associate with FGFRs to enhance the binding affinity of endocrine FGFs for their cognate FGFRs in target tissues (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007); Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Ogawa et al., Proc. Nat'l. Acad. Sci. U.S.A. 104:7432-7437 (2007); Urakawa et al., Nature 444:770-774 (2006), which are hereby incorporated by reference in their entirety). αKlotho is the co-receptor for FGF23 (Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Urakawa et al., Nature 444:770-774 (2006), which are hereby incorporated by reference in their entirety), and βKlotho is the co-receptor for both FGF19 and FGF21 (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007); Ogawa et al., Proc. Nat'l. Acad. Sci. U.S.A. 104:7432-7437 (2007), which are hereby incorporated by reference in their entirety). The C-terminal region of endocrine FGFs mediates binding of these ligands to the FGFR-α/βKlotho co-receptor complex (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007); Goetz et al., Proc. Nat'l. Acad. Sci. U.S.A. 107:407-412 (2010); Micanovic et al., J. Cell Physiol. 219:227-234 (2009); Wu et al., J. Biol. Chem. 283:33304-33309 (2008); Yie et al., FEBS Lett, 583:19-24 (2009); Goetz et al., Mol. Cell Biol. 32:1944-1954 (2012), which are hereby incorporated by reference in their entirety).
FGF23 interacts with a de novo binding site generated at the composite receptor-coreceptor interface in the binary αKlotho-FGFR complex (Goetz et al., “Isolated C-terminal Tail of FGF23 Alleviates Hypophosphatemia by Inhibiting FGF23-FGFR-Klotho Complex Formation,” Proc Natl Acad Sci USA 107:407-412 (2010), which is hereby incorporated by reference in its entirety). The region on FGF23 that binds to this de novo site was mapped to the 72 amino acid long C-terminal tail, which follows the β-trefoil core domain (Goetz et al., “Isolated C-terminal Tail of FGF23 Alleviates Hypophosphatemia by Inhibiting FGF23-FGFR-Klotho Complex Formation,” Proc Natl Acad Sci USA 107:407-412 (2010), which is hereby incorporated by reference in its entirety). Thus, the N-terminal fragment of proteolytic cleavage of FGF23 (Y25 to R179) is metabolically inactive because it lacks the binding site for the αKlotho-FGFR complex. The C-terminal proteolytic fragment (S180 to I251), however, can compete with full-length FGF23 for binding to the αKlotho-FGFR complex to antagonize the metabolic activity of FGF23, because this fragment contains the binding site for the αKlotho-FGFR complex (Goetz et al., “Isolated C-terminal Tail of FGF23 Alleviates Hypophosphatemia by Inhibiting FGF23-FGFR-Klotho Complex Formation,” Proc Natl Acad Sci USA 107:407-412 (2010), which is hereby incorporated by reference in its entirety).
Endocrine FGFs still possess residual HS-binding affinity, and moreover, there are differences in this residual binding affinity among the endocrine FGFs (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety). These observations raise the possibility that HS may play a role in endocrine FGF signaling. Indeed, there are several reports showing that HS can promote endocrine FGF signaling in the presence as well as in the absence of Klotho co-receptor. It has been shown that HS augments the mitogenic signal elicited by endocrine FGFs in BaF3 cells over-expressing FGFR and Klotho co-receptor by at least two-fold (Suzuki et al., Mol. Endocrinol. 22:1006-1014 (2008), which is hereby incorporated by reference in its entirety). In addition, even in the absence of Klotho co-receptor, HS enables endocrine FGFs to induce proliferation of BaF3 cells over-expressing FGFR (Yu et al., Endocrinology 146:4647-4656 (2005); Zhang et al., J. Biol. Chem. 281:15694-15700 (2006), which are hereby incorporated by reference in their entirety). Compared to paracrine FGFs, however, significantly higher concentrations of both ligand and HS are needed, and the proliferative response of cells to endocrine FGFs still lags behind that of paracrine FGFs by about one order of magnitude (Zhang et al., J. Biol. Chem. 281:15694-15700 (2006), which is hereby incorporated by reference in its entirety).
As used herein, the terms “chimeric polypeptide” and “chimeric protein” encompass a polypeptide having a sequence that includes at least a portion of a full-length sequence of first polypeptide sequence and at least a portion of a full-length sequence of a second polypeptide sequence, where the first and second polypeptides are different polypeptides. A chimeric polypeptide also encompasses polypeptides that include two or more non-contiguous portions derived from the same polypeptide. A chimeric polypeptide or protein also encompasses polypeptides having at least one substitution, wherein the chimeric polypeptide includes a first polypeptide sequence in which a portion of the first polypeptide sequence has been substituted by a portion of a second polypeptide sequence.
As used herein, the term “N-terminal portion” of a given polypeptide sequence is a contiguous stretch of amino acids of the given polypeptide sequence that begins at or near the N-terminal residue of the given polypeptide sequence. An N-terminal portion of the given polypeptide can be defined by a contiguous stretch of amino acids (e.g., a number of amino acid residues). Similarly, the term “C-terminal portion” of a given polypeptide sequence is a contiguous length of the given polypeptide sequence that ends at or near the C-terminal residue of the given polypeptide sequence. A C-terminal portion of the given polypeptide can be defined by a contiguous stretch of amino acids (e.g., a number of amino acid residues).
The term “portion,” when used herein with respect to a given polypeptide sequence, refers to a contiguous stretch of amino acids of the given polypeptide's sequence that is shorter than the given polypeptide's full-length sequence. A portion of a given polypeptide may be defined by its first position and its final position, in which the first and final positions each correspond to a position in the sequence of the given full-length polypeptide. The sequence position corresponding to the first position is situated N-terminal to the sequence position corresponding to the final position. The sequence of the portion is the contiguous amino acid sequence or stretch of amino acids in the given polypeptide that begins at the sequence position corresponding to the first position and ending at the sequence position corresponding to the final position. A portion may also be defined by reference to a position in the given polypeptide sequence and a length of residues relative to the referenced position, whereby the sequence of the portion is a contiguous amino acid sequence in the given full-length polypeptide that has the defined length and that is located in the given polypeptide in reference to the defined position.
As noted above, a chimeric protein according to the present invention may include an N-terminus coupled to a C-terminus. N-terminus and C-terminus are used herein to refer to the N-terminal region or portion and the C-terminal region or portion, respectively, of the chimeric protein of the present invention. In some embodiments of the present invention, the C-terminal portion and the N-terminal portion of the chimeric protein of the present invention are contiguously joined. In alternative embodiments, the C-terminal portion and the N-terminal portion of the chimeric protein of the present invention are coupled by an intervening spacer. In one embodiment, the spacer may be a polypeptide sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues. In some embodiments, the C-terminal portion and/or the N-terminal portion of the chimeric protein of the present invention may include additional portion(s) coupled to the C-terminal residue and/or the N-terminal residue of the chimeric protein of the present invention, respectively. In some embodiments, the additional portion(s) may be a polypeptide sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues. In some embodiments, the N-terminal portion and/or the C-terminal portion having such additional portion(s) will maintain the activity of the corresponding naturally occurring N-terminal portion and/or C-terminal portion, respectively. In some embodiments, the N-terminal portion and/or the C-terminal portion having such additional portion(s) will have enhanced and/or prolonged activity compared to the corresponding naturally occurring N-terminal portion and/or C-terminal portion, respectively. In other embodiments, the C-terminal portion and/or the N-terminal portion of the chimeric protein of the present invention do not include any additional portion(s) coupled to the C-terminal residue and/or the N-terminal residue of the chimeric protein of the present invention, respectively.
The portion of the paracrine FGF may be derived from any suitable paracrine FGF. Suitable paracrine FGFs in accordance with the present invention include FGF1, FGF2, and ligands of the FGF4 and FGF9 subfamilies. Certain embodiments of the present invention may include a full-length amino acid sequence of a paracrine FGF, rather than a portion of a paracrine FGF.
In one embodiment, the portion of the paracrine FGF is derived from a mammalian FGF. In one embodiment, the portion of the paracrine FGF is derived from a vertebrate FGF. In one embodiment, the portion of the paracrine FGF is derived from a human FGF. In one embodiment, the paracrine FGF is derived from a non-human mammalian FGF. In one embodiment, the portion of the paracrine FGF is derived from a non-human vertebrate FGF. In one embodiment, the paracrine FGF is derived from an ortholog of human FGF, or a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species.
In one embodiment according to the present invention, the portion of the paracrine FGF of the chimeric protein includes an N-terminal portion of the paracrine FGF.
In one embodiment, the paracrine FGF is FGF1. In one embodiment, the portion of the FGF1 is from human FGF1 having the following amino acid sequence (GenBank Accession No. AAH32697, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 1):









1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ



 


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK


 


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD






In one embodiment, the portion of the paracrine FGF includes an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 150 to 155 of SEQ ID NO: 1 (human FGF1). In one embodiment, the portion of the paracrine FGF includes amino acid residues 1-150, 1-151, 1-152, 1-153, 1-154, 1-155, 2-150, 2-151, 2-152, 2-153, 2-154, 2-155, 3-150, 3-151, 3-152, 3-153, 3-154, 3-155, 4-150, 4-151, 4-152, 4-153, 4-154, 4-155, 5-150, 5-151, 5-152, 5-153, 5-154, 5-155, 6-150, 6-151, 6-152, 6-153, 6-154, 6-155, 7-150, 7-151, 7-152, 7-153, 7-154, 7-155, 8-150, 8-151, 8-152, 8-153, 8-154, 8-155, 9-150, 9-151, 9-152, 9-153, 9-154, 9-155, 10-150, 10-151, 10-152, 10-153, 10-154, 10-155, 11-150, 11-151, 11-152, 11-153, 11-154, 11-155, 12-150, 12-151, 12-152, 12-153, 12-154, 12-155, 13-150, 13-151, 13-152, 13-153, 13-154, 13-155, 14-150, 14-151, 14-152, 14-153, 14-154, 14-155, 15-150, 15-151, 15-152, 15-153, 15-154, 15-155, 16-150, 16-151, 16-152, 16-153, 16-154, 16-155, 17-150, 17-151, 17-152, 17-153, 17-154, 17-155, 18-150, 18-151, 18-152, 18-153, 18-154, 18-155, 19-150, 19-151, 19-152, 19-153, 19-154, 19-155, 20-150, 20-151, 20-152, 20-153, 20-154, 20-155, 21-150, 21-151, 21-152, 21-153, 21-154, 21-155, 22-150, 22-151, 22-152, 22-153, 22-154, 22-155, 23-150, 23-151, 23-152, 23-153, 23-154, 23-155, 24-150, 24-151, 24-152, 24-153, 24-154, 24-155, 25-150, 25-151, 25-152, 25-153, 25-154, or 25-155 of FGF1 (SEQ ID NO: 1). In one embodiment, the portion of the paracrine FGF includes amino acid residues 1-150 or 25-150 of SEQ ID NO: 1.
In one embodiment, the portion of the paracrine FGF includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 150 to 155 of SEQ ID NO: 1 (human FGF1). In one embodiment, the portion of the paracrine FGF includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence homology to an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 150 to 155 of SEQ ID NO: 1 (human FGF1).
Percent (%) amino acid sequence identity with respect to a given polypeptide sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent (%) amino acid sequence homology with respect to a given polypeptide sequence identified herein is the percentage of amino acid residues in a candidate sequence that are identical to or strongly similar to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence homology. Strongly similar amino acid residues may include, for example, conservative amino acid substitutions known in the art. Alignment for purposes of determining percent amino acid sequence identity and/or homology can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
In one embodiment of the present invention, the portion of the paracrine FGF of the chimeric protein is derived from an ortholog of human FGF1. In one embodiment, the portion of FGF1 is derived from Papio Anubis, Pongo abelii, Callithrix jacchus, Equus caballus, Pan troglodytes, Loxodonta Africana, Canis lupus familiaris, Ailuropoda melanoleuca, Saimiri boliviensis boliviensis, Sus scrofa, Otolemur garnettii, Rhinolophus ferrumequinum, Sorex araneus, Oryctolagus cuniculus, Cricetulus griseus, Sarcophilus harrisii, Mus musculus, Cavia porcellus, Monodelphis domestica, Desmodus rotundus, Bos taurus, Ornithorhynchus anatinus, Taeniopygia guttata, Dasypus novemcinctus, Xenopus Silurana tropicalis, Heterocephalus glaber, Pteropus alecto, Tupaia chinensis, Columba livia, Ovis aries, Gallus gallus, Vicugna pacos, Anolis carolinensis, Otolemur garnettii, Felis catus, Pelodiscus sinensis, Latimeria chalumnae, Tursiops truncates, Mustela putorius furo, Nomascus leucogenys, Gorilla gorilla, Erinaceus europaeus, Procavia capensis, Dipodomys ordii, Petromyzon marinus, Echinops telfairi, Macaca mulatta, Pteropus vampyrus, Myotis lucifugus, Microcebus murinus, Ochotona princeps, Rattus norvegicus, Choloepus hoffmanni, Ictidomys tridecemlineatus, Tarsius syrichta, Tupaia belangeri, Meleagris gallopavo, Macropus eugenii, or Danio rerio. The portions of an ortholog of human paracrine FGF1 include portions corresponding to the above-identified amino acid sequences of human FGF1. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
In one embodiment, the portion of the FGF1 of the chimeric protein of the present invention is derived from an ortholog of human FGF1 having the amino acid sequence shown in Table 1.







TABLE 1







Amino acid sequence of human FGF1 (SEQ ID NO: 1) (GenBank


accession no. AAH32697, which is hereby incorporated by


reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Papio anubis (olive baboon) FGF1


(SEQ ID NO: 2) (GenBank accession no. NP_001162557,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP ANYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Pongo abelii (Sumatran orangutan) FGF1


(SEQ ID NO: 3) (GenBank accession no. NP_001127073,


which is hereby incorporated by reference in its entirety)








60
                                                                M


61
AEGEITTFTA LTEKFNLPPG NYKKPKLLYC SNGGHFLRIL PDGTVDGTRD RSDQHIQLQL


121
SAESVGEVYI KSTETGQYLA MDTDGLLYGS QTPNEECLFL ERLEENHYNT YISKKHAEKN


181
WFVGLKKNGS CKRGPRTHYG QKAILFLPLP VSSD


 







Amino acid sequence of Callithrix jacchus (white-tufted-ear


marmoset) FGF1 (SEQ ID NO: 4) (GenBank accession no.


XP_002744341, which is hereby incorporated by


reference in its entirety):








1
MAEGEITTFT ALTEKFDLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Equus caballus (horse) FGF1


(SEQ ID NO: 5) (GenBank accession no. NP_001157358,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Pan troglodytes (chimpanzee) FGF1


(SEQ ID NO: 6) (GenBank accession no. JAA29511, which is


hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPS GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Loxodonta africana (elephant) FGF1


(SEQ ID NO: 7) (GenBank accession no. XP_003404621,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKGTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Canis lupus familiaris (dog) FGF1


(SEQ ID NO: 8) (GenBank accession no. XP_849274,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYMKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Ailuropoda melanoleuca (giant panda)


FGF1 (SEQ ID NO: 9) (GenBank accession no. XP_002912581,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPA GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Saimiri boliviensis boliviensis


(Bolivian squirrel monkey) FGF1(SEQ ID NO: 10) (GenBank


accession no. XP_003920596, which is hereby


incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFDLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDLHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Sus scrofa (pig) FGF1


(SEQ ID NO: 11) (GenBank accession no. XP_003124058,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTSGLLYG SQTPSEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Otolemur garnettii (small-eared galago)


FGF1(SEQ ID NO: 12) (GenBank accession no. XP_003782135,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTQ DRSDQHIQLQ


61
LSAESVGEVY IKSTQTGQYL AMDSDGLLYG SQTPNEECLF LERLEENHYN TYVSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Rhinolophus ferrumequinum (greater


horseshoe bat) FGF1 (SEQ ID NO: 13) (GenBank accession no.


ACC62496, which is hereby incorporated by reference


in its entirety):








1
MAEGEVTTFT ALTEKFNLPT GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ


61
LSAESVGEVY IKSTESGQYL AMDSDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Sorex araneus (European shrew) FGF1


(SEQ ID NO: 14) (GenBank accession no. ACE75805, which is


hereby incorporated by reference in its entirety):








1
MAEGEITTFG ALMEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGHYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Oryctolagus cuniculus (rabbit) FGF1


(SEQ ID NO: 15) (GenBank accession no. NP_001164959,


which is hereby incorporated by reference in its entirety):








1
MAEGEVTTFT ALTEKFNLPA GNYKLPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPSEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Cricetulus griseus (Chinese hamster)


FGF1 (SEQ ID NO: 16) (GenBank accession no. XP_003502469,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFS ALTERFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESAGEVY IKGTETGQYR NMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Sarcophilus harrisii (Tasmanian devil)


FGF1 (SEQ ID NO: 17) (GenBank accession no. XP_003756738,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRNDQHIQLQ


61
LSAESVGEVY IKSTESGQYL AMDTDGLLYG SQTPTEECLF LERLEENHYN TYISKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSE


 







Amino acid sequence of Mus musculus (house mouse) FGF1


(SEQ ID NO: 18) (GenBank accession no. NP_034327,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFA ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESAGEVY IKGTETGQYL AMDTEGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Cavia porcellus (domestic guinea pig)


FGF1 (SEQ ID NO: 19) (GenBank accession no. XP_003477242,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFA ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAEGVGEVY IQSTETGQYL AMDTDGLLYG SQTPSEECLF LERLEENHYN TYTSKKHVEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSD


 







Amino acid sequence of Monodelphis domestica (gray short-tailed


opossum) FGF1(SEQ ID NO: 20) (GenBank accession no.


XP_001368921, which is hereby incorporated by


reference in its entirety):








1
MAEGEITTFT ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRNDQHIQLQ


61
LSTESVGEVY IKSTESGQYL AMDTDGLLYG SQTPSEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKKGPRTHY GQKAILFLPL PVSSE


 







Amino acid sequence of Desmodus rotundus (common vampire bat)


FGF1 (SEQ ID NO: 21) (GenBank accession no. JAA45191, which


is hereby incorporated by reference in its entirety):








1
MAEGEVTTFT ALTEKFNLPL ESYKKPKLLY CSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ


61
LSAESVGEVY IKSTGSGQYL AMDSAGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVNSD


 







Amino acid sequence of Bos taurus (cattle) FGF1 (SEQ ID NO: 22)


(GenBank accession no. NP_776480, which is hereby


incorporated by reference in its entirety):








1
MAEGETTTFT ALTEKFNLPL GNYKKPKLLY CSNGGYFLRI LPDGTVDGTK DRSDQHIQLQ


61
LCAESIGEVY IKSTETGQFL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK


121
HWFVGLKKNG RSKLGPRTHF GQKAILFLPL PVSSD


 







Amino acid sequence of Ornithorhynchus anatinus (platypus)


FGF1 (SEQ ID NO: 23) (GenBank accession no. XP_001514861,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALMEKFDLPL GNYKKPRLLY CSNGGYFLRI QPDGKVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTESGHYL AMDTEGLLYG SQAPSEDCLF LERLEENHYN TYVSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVASD


 







Amino acid sequence of Taeniopygia guttata (zebra finch) FGF1


(SEQ ID NO: 24) (GenBank accession no. XP_002193287,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFS ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGVVH IQSTQSGQYL AMDTNGLLYG SQLPPGECLF LERLEENHYN TYVSKMHADK


121
NWFVGLKKNG TSKLGPRTHY GQKAILFLPL PVAAD


 







Amino acid sequence of Dasypus novemcinctus (nine-banded


armadillo) FGF1 (SEQ ID NO: 25) (GenBank accession no.


ACO06224, which is hereby incorporated by reference


in its entirety):








1
MAEGEITTFM ALMEKFNLPL ENYKHPRLLY CRNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSAETGQYL AMDTDGLLYG SETPSEECLF MEKLEENNYN TYISKKHAEK


121
KWFVGLKKDG SSKRGPQTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Xenopus Silurana tropicalis (western


clawed frog) FGF1(SEQ ID NO: 26) (GenBank accession no.


ACJ50585, which is hereby incorporated by reference


in its entirety):








1
MAEGDITTFN PIAESFSLPI GNYKKPKLLY CNNGGYFLRI LPDGVVDGTR DRDDLYITLK


61
LSAQSQGEVH IKSTETGSYL AMDSSGQLYG TLTPNEESLF LETLEENHYN TYKSKKYAEN


121
NWFVGIKKNG ASKKGSRTHY GQKAILFLPL PASPD


 







Amino acid sequence of Heterocephalus glaber (naked mole-rat)


FGF1 (SEQ ID NO: 27) (GenBank accession no. EHA99379,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRSDQHIQLQ


61
LSAEGVGEVY IKSTETGQYL AMDTDGLLYG SQTASEECLF LERLEENHYN TYISKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Pteropus alecto (black flying fox)


FGF1 (SEQ ID NO: 28) (GenBank accession no. ELK02961,


which is hereby incorporated by reference in its entirety):








1
MAEGEVTTFT ALTERFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ


61
LSAESVGEVY IKSTESGQYL AMDSDGLLYG SQTPDEDCLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Tupaia chinensis (Chinese tree shrew)


FGF1 (SEQ ID NO: 29) (GenBank accession no. ELW69091,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFA ALTEKFDLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LTAENVGEVY IKSTETGQYL AMDADGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK


121
NWFVALKKNG SCKLGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Columba livia (rock pigeon) FGF1


(SEQ ID NO: 30) (GenBank accession no. EMC79997, which is


hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTQSGQYL AMDPTGLLYG SQLLGEECLF LERIEENHYN TYVSKKHADK


121
NWFVGLKKNG NSKLGPRTHY GQKAILFLPL PVSAD


 







Amino acid sequence of Ovis aries (sheep) FGF1 (SEQ ID NO: 31)


(GenBank accession no. XP_004008958, which is hereby


incorporated by reference in its entirety):








1
MAEGETTTFR ALTEKFNLPL GNYKKPKLLY CSNGGYFLRI LPDGRVDGTK DRSDQHIQLQ


61
LYAESIGEVY IKSTETGQFL AMDTNGLLYG SQTPSEECLF LERLEENHYN TYISKKHAEK


121
NWFIGLKKNG SSKLGPRTHF GQKAILFLPL PVSSD


 







Amino acid sequence of Gallus gallus (chicken) FGF1


(SEQ ID NO: 32) (GenBank accession no. NP_990511,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTERFGLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRSDQHIQLQ


61
LSAEDVGEVY IKSTASGQYL AMDTNGLLYG SQLPGEECLF LERLEENHYN TYISKKHADK


121
NWFVGLKKNG NSKLGPRTHY GQKAILFLPL PVSAD


 







Amino acid sequence of Vicugna pacos (alpaca) FGF1


(SEQ ID NO: 33) (Ensembl accession no. ENSVPAP00000007810;


partial sequence corresponding to human FGF1 residues 58


to 155, which is hereby incorporated by reference in


its entirety):








1
QLQLSAESVG EVYIKSTETG QYLAMDTDGL LHGSQTPNEE CLFLERLEEN HYNTYTSKKH


61
AEKNWFVGLK KNGSCKRGPR THYGQKAILF LPLPVSSD


 







Amino acid sequence of Anolis carolinensis (anole lizard) FGF1


(SEQ ID NO: 34) (Ensembl accession no. ENSACAP00000013203,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTERFALPM ENYKKPKLLY CSNGGHFLRI LPDGKVDGTM DRNDSYIQLL


61
LTAEDVGVVY IKGTETGQYL AMDANGHLYG SQLPTEECLF VETLEENHYN TYTSKMHGDK


121
KWYVGLKKNG KGKLGPRTHR GQKAILFLPL PVSPD


 







Amino acid sequence of Otolemur garnettii (bushbaby) FGF1


(SEQ ID NO: 35) (Ensembl accession no. ENSOGAP00000004540,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTQ DRSDQHIQLQ


61
LSAESVGEVY IKSTQTGQYL AMDSDGLLYG SQTPNEECLF LERLEENHYN TYVSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Felis catus (cat) FGF1(SEQ ID NO: 36)


(Ensembl accession no. ENSFCAP00000008457, which is hereby


incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Pelodiscus sinensis (Chinese


softshell turtle) FGF1 (SEQ ID NO: 37) (Ensembl accession


no. ENSPSIP00000016356, which is hereby incorporated by


reference in its entirety):








1
MAEGEITTFT ALTEKFNLPL GNYKNPKLLY CSNGGYFLRI HPDGKVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTESGQFL AMDANGLLYG SLSPSEECLF LERMEENHYN TYISKKHADK


121
NWFVGLKKNG SCKLGPRTHY GQKAVLFLPL PVSAD


 







Amino acid sequence of Latimeria chalumnae (coelacanth)


FGF1 (SEQ ID NO: 38) (Ensembl accession no. ENSLACP00000015106,


which is hereby incorporated by reference in its entirety):








1
MAEDKITTLK ALAEKFNLPM GNYKKAKLLY CSNGGYFLRI PPDGKVEGIR ERSDKYIQLQ


61
MNAESLGMVS IKGVEAGQYL AMNTNGLLYG SQSLTEECLF MEKMEENHYN TYRSKTHADK


121
NWYVGIRKNG SIKPGPRTHI GQKAVLFLPL PASSD


 







Amino acid sequence of Tursiops truncatus (dolphin) FGF1


(SEQ ID NO: 39) (Ensembl accession no. ENSTTRP00000004470,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYASKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Mustela putorius furo (ferret) FGF1


(SEQ ID NO: 40) (Ensembl accession no. ENSMPUP00000007888,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALMEKFNLPA GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Nomascus leucogenys (gibbon) FGF1


(SEQ ID NO: 41) (Ensembl accession no. ENSNLEP00000011873,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Gorilla gorilla (gorilla) FGF1


(SEQ ID NO: 42) (Ensembl accession no. ENSGGOP00000017663,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Erinaceus europaeus (hedgehog)


FGF1 (SEQ ID NO: 43) (Ensembl accession no. ENSEEUP00000005318,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Procavia capensis (hyrax) FGF1


(SEQ ID NO: 44) (Ensembl accession no. ENSPCAP00000010969,


which is hereby incorporated by reference in its entirety)


(partial sequence corresponding to human FGF1


residues 1 to 91):








1
MAEGEITTFT ALTEKFNLPL ENYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKGTETGQYL AMDTDGLLYG S


 







Amino acid sequence of Dipodomys ordii (kangaroo rat)


FGF1 (SEQ ID NO: 45) (Ensembl accession no. ENSDORP00000006889,


which is hereby incorporated by reference in its entirety)


(partial sequence corresponding to human FGF1 residues


1 to 16 and 58 to 155):








1
MAEGEITTFT ALTERF---- ---------- ---------- ---------- -------QLQ


61
LSAESVGEVY IKSTETGQYL AMDADGLLYG SQTPDEECLF LERLEENHYN TYIAKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Petromyzon marinus (lamprey) FGF1


(SEQ ID NO: 46) (Ensembl accession no. ENSPMAP00000010683,


which is hereby incorporated by reference in its entirety)


(partial sequence corresponding to human FGF1


residues 1 to 93):








1
MEVGHIGTLP VVPAGPVFPG SFKEPRRLYC RSAGHHLQIL GDGTVSGTQD ENEPHAVLQL


61
QAVRRGVVTI RGLCAERFLA MSTEGHLYGA VR


 







Amino acid sequence of Echinops telfairi (lesser hedgehog


tenrec) FGF1 (SEQ ID NO: 47) (Ensembl accession no.


ENSETEP00000014504, which is hereby incorporated by


reference in its entirety) (partial sequence corresponding to


human FGF1 residues 58 to 155)








1
QLKLVAESVG VVYIKSIKTG QYLAMNPDGL LYGSETPEEE CLFLETLEEN HYTTFKSKKH


61
VEKNWFVGLR KNGRVKIGPR THQGQKAILF LPLPVSSD


 







Amino acid sequence of Macaca mulatta (rhesus monkey) FGF1


(SEQ ID NO: 48) (Ensembl accession no. ENSMMUP00000030943,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Pteropus vampyrus (megabat) FGF1


(SEQ ID NO: 49) (Ensembl accession no. ENSPVAP00000004349,


which is hereby incorporated by reference in its entirety):








1
MAEGEVTTFT ALTERFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ


61
LSAESVGEVY IKSTESGQYL AMDSDGLLYG SQTPDEDCLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Myotis lucifugus (microbat) FGF1


(SEQ ID NO: 50) (Ensembl accession no. ENSMLUP00000006481,


which is hereby incorporated by reference in its entirety):








1
MAEGEVTTFT ALTERFNLPL ENYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTESGQYL AMDSDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Microcebus murinus (mouse lemur)


FGF1 (SEQ ID NO: 51) (Ensembl accession no. ENSMICP00000008602,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESAGEVY IKSTQTGRYL AMDADGLLYG SQTPNEECLF LERLEENHYN TYVSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Ochotona princeps (pika) FGF1


(SEQ ID NO: 52) (Ensembl accession no. ENSOPRP00000011739,


which is hereby incorporated by reference in its entirety):








1
MAEGEVTTFS ALTEKFNLPG GNYKLPKLLY CSNGGHFLRI LPDGTVDGTR DRSDLH----


61
-------EVF IKSTETGQYL AMDTDGLLYG SQTPSEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGIKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Rattus norvegicus (rat) FGF1


(SEQ ID NO: 53) (Ensembl accession no. ENSRNOP00000018577,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFA ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESAGEVY IKGTETGQYL AMDTEGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Choloepus hoffmanni (sloth) FGF1


(SEQ ID NO: 54) (Ensembl accession no. ENSCHOP00000010964,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALMEKFNLPP GNYMKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDLHIQLQ


61
LSAESVGEVY IKSAETGQYL AMDTGGLLYG SQTPSEECLF LERLEENHYN TYVSKKHAEK


121
NWFVGLKKNG SSKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Ictidomys tridecemlineatus (squirrel)


FGF1 (SEQ ID NO: 55) (Ensembl accession no. ENSSTOP00000021782,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Tarsius syrichta (tarsier) FGF1


(SEQ ID NO: 56) (Ensembl accession no. ENSTSYP00000006804,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYVSKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Tupaia belangeri (tree shrew) FGF1


(SEQ ID NO: 57) (Ensembl accession no. ENSTBEP00000010264,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFA ALTEKFDLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


61
LTAENVGEVY IKSTETGQYL AMDADGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK


121
NWFVALKKNG SCKLGPRTHY GQKAILFLPL PVSSD


 







Amino acid sequence of Meleagris gallopavo (turkey) FGF1


(SEQ ID NO: 58) (Ensembl accession no. ENSMGAP00000016398;


partial sequence corresponding to human FGF1 residues 1 to 56,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTERFGLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRSDQH


 







Amino acid sequence of Macropus eugenii (wallaby) FGF1


(SEQ ID NO: 59) (Ensembl accession no. ENSMEUP00000015084,


which is hereby incorporated by reference in its entirety):








1
MAEGEITTFT ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRNDQHIQLQ


61
LSAESVGEVY IKSTESGQYL AMDTNGLLYG SQTPSEECLF LERLEENHYN TYISKKHAEK


121
NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSE


 







Amino acid sequence of Danio rerio (zebrafish) FGF1


(SEQ ID NO: 60) (Ensembl accession no. ENSDARP00000008825,


which is hereby incorporated by reference in its entirety):








1
MTEADIAVKS SPRDYKKLTR LYCMNGGFHL QILADGTVAG AADENTYSIL RIKATSPGVV


61
VIEGSETGLY LSMNEHGKLY ASSLVTDESY FLEKMEENHY NTYQSQKHGE NWYVGIKKNG


121
KMKRGPRTHI GQKAIFFLPR QVEQEED









As noted above, the portion of the paracrine FGF may be modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. In one embodiment, the modified portion of the paracrine FGF includes one or more substitutions, additions, or deletions.
In one embodiment, the one or more substitutions are located at one or more amino acid residues of SEQ ID NO: 1 selected from N33, K127, K128, N129, K133, R134, R137, Q142, K143, and combinations thereof. In one embodiment, the one or more substitutions are selected from N33T, K127D, K128Q, N129T, K133V, R134L, R137H, Q142M, K143T/L/I, and combinations thereof. In one embodiment, the modification is one or more substitutions which are located at one or more amino acid residues corresponding to residues of SEQ ID NO: 1 selected from N33, K127, K128, N129, K133, R134, R137, Q142, K143, and combinations thereof. In one embodiment, the modification is one or more substitutions which are located at one or more amino acid residues corresponding to residues of SEQ ID NO: 1 selected from N33, K127, K128, N129, K133, R134, R137, Q142, K143, and combinations thereof. Amino acid residues corresponding to those of SEQ ID NO:1 may be determined by, for example, sequence analysis and structural analysis.
Also encompassed within the present invention are portions of paracrine FGFs other than FGF1 (e.g., FGF2, FGF4, FGF5, FGF6, FGF9, FGF16, and FGF20). The portions derived from paracrine FGFs other than FGF1 include portions corresponding to the above-identified amino acid sequences of FGF1. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
It will be understood that the portion of the paracrine FGF according to the present invention may be derived from a nucleotide sequence that encodes a paracrine FGF protein. For example, in one embodiment, the nucleotide sequence is the nucleotide sequence that encodes human FGF1 (GenBank Accession No. BC032697, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 61), as follows:









91
                                 ATGGCTGAAG GGGAAATCAC CACCTTCACA



 


121
GCCCTGACCG AGAAGTTTAA TCTGCCTCCA GGGAATTACA AGAAGCCCAA ACTCCTCTAC


 


181
TGTAGCAACG GGGGCCACTT CCTGAGGATC CTTCCGGATG GCACAGTGGA TGGGACAAGG


 


241
GACAGGAGCG ACCAGCACAT TCAGCTGCAG CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT


 


301
ATAAAGAGTA CCGAGACTGG CCAGTACTTG GCCATGGACA CCGACGGGCT TTTATACGGC


 


361
TCACAGACAC CAAATGAGGA ATGTTTGTTC CTGGAAAGGC TGGAGGAGAA CCATTACAAC


 


421
ACCTATATAT CCAAGAAGCA TGCAGAGAAG AATTGGTTTG TTGGCCTCAA GAAGAATGGG


 


481
AGCTGCAAAC GCGGTCCTCG GACTCACTAT GGCCAGAAAG CAATCTTGTT TCTCCCCCTG


 


541
CCAGTCTCTT CTGATTAA






In another embodiment of the present invention, the portion of the paracrine FGF of the chimeric protein may be derived from a nucleotide sequence that encodes an ortholog of human FGF1. Nucleotide sequences that encode FGF1 orthologs are shown in Table 2.







TABLE 2







Olive Baboon FGF1 gene coding sequence (1-155) (SEQ ID NO: 62)


(GenBank accession no. NM_001169086, which is hereby


incorporated by reference in its entirety):








1
ATGGCTGAAG GGGAAATCAC CACGTTCACA GCCCTGACCG AGAAGTTTAA TCTGCCTCCA


61
GCGAATTACA AGAAGCCCAA ACTGCTCTAC TGTAGCAACG GGGGACACTT CTTGAGGATC


121
CTTCCGGATG GCACAGTGGA TGGGACAAGG GACAGGAGCG ACCAGCACAT TCAGCTGCAG


181
CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CCGAGACTGG CCAGTACTTG


241
GCCATGGACA CCGACGGGCT TTTATACGGC TCACAGACAC CAAATGAGGA ATGTTTGTTC


301
CTGGAAAGGC TGGAGGAGAA CCATTACAAC ACCTACATAT CCAAGAAGCA CGCAGAGAAG


361
AATTGGTTTG TTGGCCTCAA GAAGAATGGA AGCTGCAAAC GTGGTCCTCG GACTCACTAT


421
GGCCAGAAAG CAATCTTGTT TCTTCCCCTG CCAGTCTCTT CTGATTAA


 







Sumatran orangutan FGF1 gene coding sequence (60-214) (SEQ ID NO: 63)


(GenBank accession no. NM_001133601, which is hereby


incorporated by reference in its entirety):








211
                                 ATGGCTGAAG GGGAAATCAC CACCTTCACA


241
GCCCTGACCG AGAAGTTTAA TCTGCCTCCA GGGAATTACA AGAAGCCCAA ACTCCTCTAC


301
TGTAGCAACG GGGGCCACTT CTTGAGGATC CTTCCGGATG GCACAGTGGA TGGGACAAGG


361
GACAGGAGCG ACCAGCACAT TCAGCTGCAG CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT


421
ATAAAGAGTA CCGAGACTGG CCAGTACTTG GCCATGGACA CCGACGGGCT TTTATACGGC


481
TCACAGACAC CAAATGAGGA ATGTTTGTTC CTGGAAAGGC TGGAGGAGAA CCATTACAAC


541
ACCTATATAT CCAAGAAGCA TGCAGAGAAG AATTGGTTTG TTGGCCTCAA GAAGAATGGA


601
AGCTGCAAAC GCGGTCCTCG GACTCACTAT GGCCAGAAAG CAATCTTGTT TCTCCCCCTG


661
CCAGTCTCTT CCGATTAA


 







White-tufted-ear marmoset FGF1 gene coding sequence (1-155)


(SEQ ID NO: 64) (GenBank accession no. XM_002744295,


which is hereby incorporated by reference in its entirety):








130
         A TGGCTGAAGG GGAAATCACC ACCTTCACAG CCCTGACCGA GAAGTTTGAT


181
CTGCCTCCAG GGAATTACAA GAAGCCCAAA CTCCTCTACT GTAGCAATGG GGGCCACTTC


241
TTGAGGATCC TTCCGGATGG CACAGTGGAT GGGACAAGGG ACAGGAGCGA CCAGCACATT


301
CAGCTGCAGC TCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTAC CGAGACTGGC


361
CAGTACTTGG CCATGGACAC CGACGGGCTT TTATACGGCT CACAGACACC AAATGAGGAA


421
TGTTTGTTCC TGGAGAGGCT GGAGGAGAAC CATTACAACA CCTATATATC CAAGAAACAT


481
GCAGAGAAGA ATTGGTTTGT CGGCCTCAAG AAGAATGGAA GCTGTAAACG TGGTCCTCGG


541
ACTCACTATG GTCAGAAAGC GATCTTGTTT CTCCCCCTGC CAGTTTCTTC TGATTAA


 







Horse FGF1 gene coding sequence (1-155) (SEQ ID NO: 65)


(GenBank accession no. NM_001163886, which is hereby


incorporated by reference in its entirety):








34
                                    ATGGCTG AAGGAGAAAT CACAACCTTC


61
ACGGCCCTGA CCGAGAAGTT TAATCTGCCT CCAGGGAATT ACAAGAAGCC CAAACTCCTC


121
TACTGTAGCA ATGGGGGCCA CTTCCTGAGG ATCCTTCCAG ATGGCACAGT GGATGGGACA


181
AGGGACAGGA GCGACCAGCA CATTCAGCTG CAGCTCAGTG CGGAAAGCGT GGGGGAGGTG


241
TATATAAAGA GTACCGAGAC TGGCCAGTAC TTGGCCATGG ACACCGACGG GCTGTTGTAC


301
GGCTCACAGA CACCAAACGA GGAATGTTTG TTCCTGGAAA GGCTGGAGGA AAACCATTAC


361
AACACCTACA CATCCAAGAA GCATGCAGAG AAGAACTGGT TCGTTGGTCT CAAGAAGAAT


421
GGGAGCTGCA AACGCGGTCC TCGGACTCAC TATGGGCAGA AAGCAATCTT GTTTCTTCCC


481
CTGCCCGTCT CCTCTGACTA A


 







Chimpanzee FGF1 gene coding sequence (1-155) (SEQ ID NO: 66) (GenBank


accession no. GABD01003589, which is hereby incorporated by reference


in its entirety):








80
                    A TGGCTGAAGG GGAAATCACC ACCTTCACAG CCCTGACCGA


121
GAAGTTTAAT CTGCCTTCAG GGAATTACAA GAAGCCCAAA CTCCTCTACT GTAGCAACGG


181
GGGCCACTTC CTGAGGATCC TTCCGGATGG CACAGTGGAT GGGACAAGGG ACAGGAGCGA


241
CCAGCACATT CAGCTGCAGC TCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTAC


301
CGAGACTGGC CAGTACTTGG CCATGGACAC CGACGGGCTT TTATACGGCT CACAGACACC


361
AAATGAGGAA TGTTTGTTCC TGGAACGGCT GGAGGAGAAC CATTACAACA CCTATATATC


421
CAAGAAGCAT GCAGAGAAGA ATTGGTTTGT TGGCCTCAAG AAGAATGGAA GCTGCAAACG


481
CGGTCCTCGG ACTCACTATG GCCAGAAAGC AATCTTGTTT CTCCCCCTGC CAGTCTCTTC


541
CGATTAA


 







Elephant FGF1 gene coding sequence (1-155) (SEQ ID NO: 67) (GenBank


accession no. XM_003404573, which is hereby incorporated by reference in


its entirety):








1
ATGGCCGAAG GGGAAATCAC AACTTTCACA GCCCTGACAG AGAAGTTCAA CCTGCCTCCA


61
GGGAATTACA AGAAGCCCAA ACTCCTCTAC TGTAGCAATG GAGGTCACTT CTTAAGGATC


121
CTTCCAGATG GCACAGTGGA TGGCACCAGG GACAGGAGTG ACCAGCACAT TCAGCTGCAG


181
CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGGGCA CCGAGACTGG CCAGTACTTG


241
GCCATGGACA CCGACGGGCT TTTATACGGC TCACAGACAC CAAATGAGGA ATGTTTGTTC


301
CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCA CGCAGAGAAG


361
AATTGGTTCG TTGGTCTCAA GAAGAATGGA AGCTGCAAAC GCGGTCCTCG GACTCACTAT


421
GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTCTCCT CTGATTAA


 







Dog FGF1 gene coding sequence (1-155) (SEQ ID NO: 68) (GenBank accession


no. XM_844181, which is hereby incorporated by reference in its


entirety):








164
                                               ATGGCTG AAGGGGAAAT


181
CACAACCTTC ACTGCCCTGA CGGAGAAGTT TAATCTGCCT CCGGGGAATT ACATGAAGCC


241
CAAACTCCTC TACTGTAGCA ACGGGGGCCA CTTCCTGAGG ATCCTTCCAG ATGGCACAGT


301
GGATGGGACA AGGGACAGGA GCGACCAGCA CATTCAGCTG CAGCTCAGCG CGGAAAGCGT


361
GGGGGAGGTG TATATAAAGA GCACCGAGAC TGGCCAGTAC TTGGCCATGG ACACCGATGG


421
GCTTCTGTAC GGCTCACAGA CACCGAATGA GGAATGTTTG TTCCTGGAAA GGCTGGAGGA


481
AAACCATTAC AACACCTACA CATCCAAGAA GCATGCAGAA AAAAATTGGT TTGTTGGTCT


541
CAAGAAGAAT GGAAGCTGCA AACGCGGTCC TCGGACTCAC TATGGTCAAA AAGCAATTTT


601
GTTTCTCCCC CTGCCAGTGT CCTCTGATTA A


 







Giant panda FGF1 gene coding sequence (1-155) (SEQ ID NO: 69) (GenBank


accession no. XM_002912535, which is hereby incorporated by reference in


its entirety):








146
                           ATGGC TGAAGGGGAG ATCACAACCT TCACCGCCCT


181
GACGGAGAAG TTTAATCTGC CTGCGGGGAA TTACAAGAAG CCCAAACTCC TCTACTGTAG


241
CAACGGGGGC CACTTCCTGA GGATCCTTCC AGATGGCACA GTGGACGGGA CGAGGGACAG


301
GAGCGACCAG CACATTCAAC TGCAGCTCAG CGCGGAAAGC GTAGGGGAGG TGTACATAAA


361
GAGCACCGAG ACCGGCCAGT ACTTGGCCAT GGACACCGAT GGGCTTCTGT ACGGCTCACA


421
GACACCAAAT GAGGAATGTT TGTTCCTGGA AAGGCTGGAG GAAAACCATT ACAACACCTA


481
CACATCCAAG AAGCACGCGG AGAAGAATTG GTTTGTTGGT CTCAAGAAGA ATGGAAGCTG


541
CAAACGTGGT CCTCGGACTC ACTATGGCCA GAAAGCAATT CTGTTTCTCC CCCTGCCAGT


601
CTCCTCTGAT TAA


 







Bolivian squirrel monkey FGF1 gene coding sequence (1-155)


(SEQ ID NO: 70) (GenBank accession no. XM_003920547,


which is hereby incorporated by reference in its entirety):








130
         A TGGCTGAAGG GGAAATCACC ACCTTTACAG CCCTGACCGA GAAGTTTGAT


181
CTGCCTCCAG GGAATTACAA GAAGCCCAAA CTCCTCTACT GTAGCAACGG GGGCCACTTC


241
TTGAGGATCC TTCCGGATGG CACAGTGGAT GGGACCAGGG ACAGGAGCGA TCTTCACATT


301
CAGCTGCAGC TCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTAC CGAGACTGGC


361
CAGTACTTGG CCATGGACAC CGACGGGCTT TTATACGGCT CACAGACACC AAATGAGGAA


421
TGTTTGTTCC TGGAAAGGCT GGAGGAGAAC CATTACAACA CCTATATATC CAAGAAACAC


481
GCAGAGAAGA ATTGGTTTGT TGGCCTCAAG AAGAATGGAA GCTGCAAGCG CGGTCCTCGG


541
ACTCACTATG GCCAGAAAGC AATCTTGTTT CTCCCCCTGC CAGTCTCTTC TGATTAA


 







Pig FGF1 gene coding sequence (1-155) (SEQ ID NO: 71) (GenBank accession


no. XM_003124010, which is hereby incorporated by reference in its


entirety):








35
                                     ATGGCT GAAGGCGAAA TCACAACCTT


61
CACGGCCCTG ACCGAGAAGT TTAATCTGCC TCCAGGAAAT TACAAGAAGC CCAAGCTCCT


121
CTACTGCAGC AACGGGGGCC ATTTCCTCAG GATCCTTCCA GATGGCACAG TGGATGGGAC


181
CAGGGACAGG AGCGACCAGC ACATTCAGCT GCAGCTCAGT GCGGAAAGCG TGGGGGAGGT


241
GTATATAAAG AGTACGGAGA CTGGCCAGTA CTTGGCCATG GACACCAGCG GGCTTTTGTA


301
CGGCTCACAG ACACCCAGTG AGGAGTGTTT GTTCCTGGAG AGGCTGGAGG AAAACCATTA


361
CAATACCTAC ACATCCAAGA AGCACGCAGA GAAGAACTGG TTCGTTGGCC TCAAGAAGAA


421
TGGAAGCTGC AAACGCGGTC CTCGGACTCA CTATGGCCAG AAAGCCATCC TGTTTCTCCC


481
CCTGCCAGTA TCCTCGGATT AA


 







Small-eared galago FGF1 gene coding sequence (1-155) (SEQ ID NO: 72)


(GenBank accession no. XM_003782087, which is hereby incorporated by


reference in its entirety):








28
                             ATG GCTGAAGGGG AAATCACAAC CTTCACAGCC


61
CTCACAGAGA AGTTTAATCT GCCTCTAGGA AATTACAAGA AGCCCAAGCT CCTCTACTGT


121
AGCAACGGGG GTCACTTTCT GAGGATCCTG CCGGATGGCA CCGTGGATGG GACACAAGAC


181
AGGAGCGACC AGCACATTCA GCTGCAGCTC AGTGCGGAAA GCGTGGGGGA GGTGTATATA


241
AAGAGTACCC AGACTGGCCA GTACTTGGCC ATGGACTCCG ACGGGCTTTT ATACGGCTCA


301
CAAACACCAA ATGAGGAATG CCTGTTCCTG GAACGGCTGG AGGAAAACCA TTACAACACC


361
TATGTGTCCA AGAAGCACGC CGAGAAGAAT TGGTTTGTCG GTCTCAAGAA GAACGGAAGT


421
TGCAAACGTG GTCCTCGGAC TCACTACGGC CAGAAAGCAA TCTTGTTTCT CCCCCTGCCA


481
GTCTCCTCTG ATTAA


 







Greater horseshoe bat FGF1 gene coding sequence (1-155) (SEQ ID NO: 73)


(GenBank accession no. DP000705, which is hereby incorporated


by reference in its entirety):








190120
                                          T TAATCAGAGG AGACTGGCAG


190141
GGGGAGAAAC AGGATTGCTT TCTGGCCATA GTGAGTCCGA GGACCGCGCT TGCAGCTTCC


190201
ATTCTTCTTG AGCCCAACGA ACCAATTCTT TTCTGCGTGC TTCTTGGACG TGTAGGTGTT


190261
GTAATGGTTT TCCTCCAGCC TTTCCAGGAA CAGACATTCC TCATTTGGTG TCTG


194466
     TGAGC CGTACAAAAG CCCGTCGGAG TCCATGGCCA AGTACTGGCC ACTCTCGGTG


194521
CTCTTTATAT ACACCTCCCC CACGCTTTCC GCACTGAGCT GCAGCTGAA


208114
                                    TGTGCTG GTCACTCTTG TCCCTTGTCC


208141
CATCCACTGT GCCATCTGGA AGGATCCTCA GGAAGTGGCC CCCGTTGCTG CAGTAGAGAA


208201
GTTTGGGTTT CTTGTAATTC CCTGTAGGCA GATTAAACTT CTCAGTAAGG GCTGTGAACG


208261
TGGTGACTTC CCCTTCGGCC AT


 







European shrew FGF1 gene coding sequence (1-155) (SEQ ID NO: 74)


(GenBank accession no. DP000767, which is hereby incorporated


by reference in its entirety):








138344
                                               CTAGTCG GAGGAGACGG


138361
GCAGGGGGAG AAACAAGATC GCTTTCTGGC CGTAGTGAGT CCGGGGACCA CGCTTGCAGC


138421
TTCCGTTCTT CTTCAGACCA ACAAACCAAT TCTTCTCGGC ATGCTTCTTG GAGGTATAGG


138481
TGTTGTAATG GTTTTCCTCC AGCCTTTCCA GAAACAGACA TTCCTCATTC GGTGTTTG


143512
                                                        TGAGCCGTA


143521
TAAAAGCCCG TCGGTGTCCA TGGCCAAGTA ATGGCCAGTC TCCGTGCTCT TTATATACAC


143581
CTCCCCCACG CTTTCCGCAC TGAGCTGCAG CTGAA


157009
                                                    TG TGCTGGTCGC


157021
TGCGGTCCCT GGTCCCATCC ACTGTGCCGT CCGGGAGGAT GCGCAGGAAG TGGCCCCCGT


157081
TGCTGCAGTA CAGGAGTTTG GGCTTCTTGT AGTTCCCTGG TGGCAGGTTA AACTTCTCCA


157141
TGAGGGCCCC AAAGGTGGTG ATCTCCCCCT CGGCCAT


 







Rabbit FGF1 gene coding sequence (1-155) (SEQ ID NO: 75) (GenBank


accession no. NM_001171488, which is hereby incorporated by reference in


its entirety):








1
ATGGCTGAGG GGGAGGTCAC CACCTTCACA GCCCTGACCG AGAAGTTCAA CCTGCCTGCA


61
GGGAACTACA AGTTGCCCAA ACTCCTCTAC TGCAGCAACG GGGGCCACTT CCTGAGGATC


121
CTGCCGGACG GCACTGTGGA CGGCACAAGG GACAGGAGCG ACCAGCACAT TCAGCTGCAG


181
CTGAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CGGAGACCGG CCAGTACTTG


241
GCCATGGACA CCGACGGCCT TTTATACGGC TCGCAAACGC CCAGTGAGGA GTGTTTGTTC


301
CTGGAACGGC TGGAGGAGAA CCACTACAAC ACCTACACGT CCAAGAAGCA CGCCGAGAAG


361
AACTGGTTCG TGGGGCTGAA GAAAAACGGG AGCTGCAAGC GCGGTCCTCG GACTCACTAC


421
GGCCAGAAAG CCATCTTGTT CCTCCCCCTG CCGGTCTCCT CCGACTAA


 







Chinese hamster FGF1 gene coding sequence (1-155) (SEQ ID NO: 76)


(GenBank accession no. XM_003502421, which is hereby


incorporated by reference in its entirety):








1
ATGGCTGAAG GAGAAATCAC CACCTTCTCA GCCCTGACAG AGAGATTTAA TCTGCCTCCA


61
GGAAACTACA AGAAGCCCAA ACTGCTCTAC TGCAGCAACG GGGGCCACTT CTTGAGGATC


121
CTTCCAGATG GCACAGTGGA TGGGACAAGG GACAGGAGTG ACCAGCACAT TCAGCTGCAG


181
CTGAGTGCGG AAAGCGCGGG CGAAGTGTAT ATAAAGGGTA CAGAGACAGG CCAGTACAGG


241
AACATGGACA CGGATGGCCT TTTATACGGC TCACAGACAC CAAATGAAGA ATGCCTGTTC


301
CTGGAAAGGC TGGAAGAAAA CCATTACAAC ACTTATACAT CCAAGAAGCA CGCAGAGAAG


361
AACTGGTTTG TGGGCCTCAA GAAAAACGGG AGCTGCAAGC GTGGTCCTCG GACTCACTAT


421
GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCTGTATCTT CTGACTAG


 







Tasmanian devil FGF1 gene coding sequence (1-155) (SEQ ID NO: 77)


(GenBank accession no. XM_003756690, which is hereby


incorporated by reference in its entirety):








24
                         ATGGCCG AAGGGGAGAT CACAACCTTC ACAGCCCTGA


61
CCGAAAGATT TAATCTGCCA CTGGGGAATT ACAAGAAGCC CAAGCTTCTC TACTGTAGCA


121
ATGGGGGCCA CTTTTTGAGG ATTCTTCCTG ATGGTAAAGT GGATGGGACA AGGGACAGAA


181
ATGATCAACA CATTCAACTG CAACTAAGCG CGGAAAGCGT GGGTGAGGTG TATATAAAGA


241
GCACTGAGTC TGGCCAGTAT TTGGCTATGG ACACCGATGG ACTTTTATAC GGCTCACAGA


301
CACCCACTGA AGAATGCTTG TTCCTGGAGA GATTGGAGGA GAATCATTAC AACACCTACA


361
TATCAAAGAA GCATGCGGAG AAAAATTGGT TTGTGGGCCT CAAGAAAAAT GGAAGCTGCA


421
AAAGAGGTCC CAGGACTCAC TATGGCCAGA AAGCCATCCT CTTCCTTCCC CTCCCTGTGT


481
CCTCTGAGTA A


 







House mouse FGF1 gene coding sequence (1-155) (SEQ ID NO: 78)


(GenBank accession no. NM_010197, which is hereby


incorporated by reference in its entirety):








188
       ATG GCTGAAGGGG AGATCACAAC CTTCGCAGCC CTGACCGAGA GGTTCAACCT


241
GCCTCTAGGA AACTACAAAA AGCCCAAACT GCTCTACTGC AGCAACGGGG GCCACTTCTT


301
GAGGATCCTT CCTGATGGCA CCGTGGATGG GACAAGGGAC AGGAGCGACC AGCACATTCA


361
GCTGCAGCTC AGTGCGGAAA GTGCGGGCGA AGTGTATATA AAGGGTACGG AGACCGGCCA


421
GTACTTGGCC ATGGACACCG AAGGGCTTTT ATACGGCTCG CAGACACCAA ATGAGGAATG


481
TCTGTTCCTG GAAAGGCTGG AAGAAAACCA TTATAACACT TACACCTCCA AGAAGCATGC


541
GGAGAAGAAC TGGTTTGTGG GCCTCAAGAA GAACGGGAGC TGTAAGCGCG GTCCTCGGAC


601
TCACTATGGC CAGAAAGCCA TCTTGTTTCT GCCCCTCCCG GTGTCTTCTG ACTAG


 







Domestic guinea pig FGF1 gene coding sequence (1-154) (SEQ ID NO: 79)


(GenBank accession no. XM_003477194, which is hereby incorporated by


reference in its entirety):








1
ATGGCTGAAG GAGAAATCAC AACTTTTGCA GCCCTGACTG AGAAGTTTAA TCTGCCTCCA


61
GGGAATTATA AGAAGCCCAA ACTGCTCTAC TGCAGCAATG GGGGCCACTT CCTGAGGATC


121
CTTCCAGACG GCACAGTGGA CGGCACAAGA GACAGGAGCG ACCAGCACAT TCAGCTGCAG


181
CTCAGTGCGG AAGGCGTGGG GGAGGTGTAT ATACAGAGCA CCGAGACCGG CCAGTACTTG


241
GCCATGGACA CCGACGGGCT TTTATACGGC TCACAGACAC CAAGTGAGGA ATGCTTGTTC


301
CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCA TGTGGAGAAG


361
AATTGGTTTG TTGGCCTCAA GAAGAACGGA AGCTGCAAGC GTGGTCCTCG GACTCACTAT


421
GGCCAGAAAG CAATCTTGTT CCTCCCCTTG CCAGTCTCTG ATTAG


 







Gray short-tailed opossum FGF1 gene coding sequence (1-155)


(SEQ ID NO: 80) (GenBank accession no. XM_001368884,


which is hereby incorporated by reference in its entirety):








1
ATGGCCGAAG GGGAGATCAC AACCTTCACA GCCCTGACTG AAAGATTTAA CCTGCCACTG


61
GGGAATTACA AGAAACCCAA GCTTCTCTAC TGTAGCAATG GGGGCCATTT CTTGAGGATC


121
CTTCCTGATG GCAAAGTGGA TGGGACACGG GACAGAAATG ATCAACACAT TCAACTGCAG


181
CTGAGCACGG AAAGTGTGGG TGAGGTGTAT ATAAAGAGCA CTGAGTCTGG CCAGTATTTG


241
GCTATGGACA CCGATGGACT TTTATATGGC TCACAGACAC CCAGTGAAGA ATGCTTGTTT


301
CTGGAGAGGT TGGAGGAGAA TCATTACAAC ACCTACACAT CGAAGAAGCA TGCAGAGAAA


361
AATTGGTTTG TTGGTCTCAA GAAGAATGGA AGCTGCAAAA AGGGTCCCAG GACTCACTAC


421
GGCCAGAAAG CCATCCTGTT CCTTCCCCTC CCTGTGTCCT CTGAGTAA


 







Common vampire bat FGF1 gene coding sequence (1-155) (SEQ ID NO: 81)


(GenBank accession no. GABZ01008334, which is hereby incorporated by


reference in its entirety):








1
ATGGCTGAAG GGGAAGTCAC CACGTTCACA GCTCTGACTG AGAAGTTTAA TCTGCCTCTG


61
GAGAGTTACA AGAAGCCCAA ACTTCTCTAC TGCAGCAACG GTGGCCACTT CCTGAGGATC


121
CTTCCAGATG GTACAGTGGA TGGGACAAGG GACAAGAGCG ACCAGCACAT TCAGCTGCAG


181
CTCAGTGCGG AAAGCGTGGG GGAGGTGTAC ATAAAGAGCA CCGGGAGTGG CCAGTACTTG


241
GCCATGGACT CCGCCGGGCT TTTGTATGGC TCACAGACAC CAAATGAGGA ATGTTTGTTC


301
CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCA TGCAGAAAAG


361
AATTGGTTCG TGGGGCTCAA GAAGAATGGA AGCTGCAAGC GTGGCCCCCG GACTCATTAT


421
GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTCAACT CTGATTAA


 







Cattle FGF1 gene coding sequence (1-155) (SEQ ID NO: 82) (GenBank


accession no. NM_174055, which is hereby incorporated by


reference in its entirety):








918
                  ATG GCTGAAGGAG AAACCACGAC CTTCACGGCC CTGACTGAGA


961
AGTTTAACCT GCCTCTAGGC AATTACAAGA AGCCCAAGCT CCTCTACTGC AGCAACGGGG


1021
GCTACTTCCT GAGAATCCTC CCAGATGGCA CAGTGGATGG GACGAAGGAC AGGAGCGACC


1081
AGCACATTCA GCTGCAGCTC TGTGCGGAAA GCATAGGGGA GGTGTATATT AAGAGTACGG


1141
AGACTGGCCA GTTCTTGGCC ATGGACACCG ACGGGCTTTT GTACGGCTCA CAGACACCCA


1201
ATGAGGAATG TTTGTTCCTG GAAAGGTTGG AGGAAAACCA TTACAACACC TACATATCCA


1261
AGAAGCATGC AGAGAAGCAT TGGTTCGTTG GTCTCAAGAA GAACGGAAGG TCTAAACTCG


1321
GTCCTCGGAC TCACTTCGGC CAGAAAGCCA TCTTGTTTCT CCCCCTGCCA GTCTCCTCTG


1381
ATTAA


 







Platypus FGF1 gene coding sequence (1-155) (SEQ ID NO: 83) (GenBank


accession no. XM_001514811, which is hereby incorporated


by reference in its entirety):








1
ATGGCGGAGG GTGAAATCAC CACGTTCACA GCCCTGATGG AGAAGTTCGA CCTACCCCTG


61
GGCAACTACA AAAAGCCTAG GCTGCTCTAC TGCAGCAATG GCGGCTACTT CCTGCGCATC


121
CAGCCAGACG GTAAAGTGGA CGGGACCAGG GATCGGAGCG ATCAGCACAT TCAACTGCAG


181
CTAAGCGCGG AAAGCGTGGG CGAGGTGTAT ATAAAGAGCA CCGAGTCTGG CCACTATTTG


241
GCTATGGACA CCGAAGGACT TTTATATGGC TCACAGGCAC CCAGTGAAGA CTGCTTGTTC


301
CTGGAGCGGC TGGAGGAGAA CCACTATAAC ACGTACGTGT CCAAGAAGCA CGCTGAGAAG


361
AATTGGTTTG TCGGTCTCAA GAAGAACGGG AGCTGCAAAC GAGGTCCCCG GACTCACTAC


421
GGCCAGAAAG CCATCCTCTT CCTCCCGCTC CCCGTGGCAT CCGACTAG


 







Zebra finch FGF1 gene coding sequence (1-155) (SEQ ID NO: 84) (GenBank


accession no. XM_002193251, which is hereby incorporated


by reference in its entirety):








1
ATGGCCGAGG GGGAGATCAC CACCTTCAGC GCCCTGACGG AGAAGTTCAA CCTGCCCCCG


61
GGGAACTACA AGAAGCCCAA ACTGCTGTAC TGCAGCAACG GGGGGCATTT CCTGCGCATC


121
CTCCCGGACG GCACCGTGGA TGGCACCAGG GACCGCAGCG ACCAGCACAT TCAGCTCCAG


181
CTGAGTGCAG AGAGCGTGGG GGTGGTGCAC ATCCAGAGCA CCCAGTCGGG GCAGTACCTG


241
GCCATGGACA CCAACGGGCT GCTCTACGGC TCGCAGCTGC CACCCGGTGA GTGTCTGTTC


301
CTGGAAAGGC TGGAGGAGAA CCATTACAAC ACCTACGTCT CCAAAATGCA CGCGGACAAG


361
AACTGGTTTG TGGGGCTGAA GAAGAACGGG ACAAGCAAGC TGGGCCCGCG GACTCACTAC


421
GGCCAGAAGG CGATCCTGTT CCTGCCGCTG CCCGTGGCGG CCGACTGA


 







Nine-banded armadillo FGF1 gene coding sequence (1-155) (SEQ ID NO: 85)


(GenBank accession no. DP001080, which is hereby incorporated by


reference in its entirety):








178389
        TT AATCAGAGGA GACTGGCAGG GGAAGAAACA AGATAGCTTT CTGGCCATAG


178441
TGAGTCTGAG GACCACGTTT GCTGCTTCCG TCCTTCTTGA GACCAACAAA CCATTTCTTC


178501
TCTGCATGCT TCTTGGATAT GTAGGTGTTG TAATTGTTTT CTTCCAGCTT TTCCATGAAC


178561
AAGCATTCCT CACTTGGTGT CTC


182873
                                                         TGAGCCAT


182881
ATAAAAGCCC GTCGGTGTCC ATGGCTAAGT ACTGGCCGGT CTCTGCACTC TTTATATACA


182941
CCTCCCCCAC GCTTTCCGCA CTGAGCTGCA GCTGAA


197786
                           TGTGT TGGTCGCTCC TGTCCCTTGT CCCATCCACC


197821
GTGCCATCTG GAAGGATCCT CAAGAAGTGG CCCCCGTTTC TGCAGTAGAG GAGTCTGGGG


197881
TGCTTGTAAT TTTCTAGGGG CAGGTTGAAC TTCTCCATCA GGGCCATGAA GGTTGTGATC


197941
TCCCCTTCAG CCAT


 







Xenopus Silurana tropicalis FGF1 gene coding sequence (1-155)


(SEQ ID NO: 86) (GenBank accession no. FJ428265,


which is hereby incorporated by reference in its entirety):








1
ATGGCAGAGG GAGACATCAC AACATTCAAC CCCATTGCAG AGTCCTTCAG TCTTCCAATT


61
GGCAACTACA AGAAACCAAA ACTTCTGTAC TGTAATAATG GAGGGTATTT TTTGCGCATC


121
CTCCCAGATG GGGTTGTGGA TGGAACAAGA GACAGAGATG ACCTTTACAT TACACTGAAG


181
TTAAGCGCAC AAAGCCAAGG GGAGGTGCAT ATCAAAAGCA CAGAGACAGG GAGTTACTTA


241
GCCATGGACT CCAGTGGACA GTTGTATGGA ACTCTCACAC CAAATGAAGA AAGCCTGTTT


301
CTGGAGACAT TAGAAGAGAA TCACTATAAC ACATACAAGT CAAAGAAGTA TGCAGAAAAT


361
AACTGGTTTG TGGGGATAAA GAAGAACGGG GCAAGCAAAA AGGGATCAAG GACTCACTAT


421
GGACAAAAAG CCATCCTTTT TCTGCCGCTG CCAGCATCAC CTGACTAG


 







Heterocephalus glaber FGF1 gene coding sequence (1-155) (SEQ ID NO: 87)


(generated using SMS Reverse Translate tool on the ExPASy Bioinformatics


Resource website (www.expasy.org):








1
ATGGCGGAAG GCGAAATTAC CACCTTTACC GCGCTGACCG AAAAATTTAA CCTGCCGCCG


61
GGCAACTATA AAAAACCGAA ACTGCTGTAT TGCAGCAACG GCGGCCATTT TCTGCGCATT


121
CTGCCGGATG GCAAAGTGGA TGGCACCCGC GATCGCAGCG ATCAGCATAT TCAGCTGCAG


181
CTGAGCGCGG AAGGCGTGGG CGAAGTGTAT ATTAAAAGCA CCGAAACCGG CCAGTATCTG


241
GCGATGGATA CCGATGGCCT GCTGTATGGC AGCCAGACCG CGAGCGAAGA ATGCCTGTTT


301
CTGGAACGCC TGGAAGAAAA CCATTATAAC ACCTATATTA GCAAAAAACA TGCGGAAAAA


361
AACTGGTTTG TGGGCCTGAA AAAAAACGGC AGCTGCAAAC GCGGCCCGCG CACCCATTAT


421
GGCCAGAAAG CGATTCTGTT TCTGCCGCTG CCGGTGAGCA GCGAT


 







Black flying fox FGF1 gene coding sequence (1-155) (SEQ ID NO: 88)


(generated using SMS Reverse Translate tool on the ExPASy Bioinformatics


Resource website (www.expasy.org):








1
ATGGCGGAAG GCGAAGTGAC CACCTTTACC GCGCTGACCG AACGCTTTAA CCTGCCGCCG


61
GGCAACTATA AAAAACCGAA ACTGCTGTAT TGCAGCAACG GCGGCCATTT TCTGCGCATT


121
CTGCCGGATG GCACCGTGGA TGGCACCCGC GATAAAAGCG ATCAGCATAT TCAGCTGCAG


181
CTGAGCGCGG AAAGCGTGGG CGAAGTGTAT ATTAAAAGCA CCGAAAGCGG CCAGTATCTG


241
GCGATGGATA GCGATGGCCT GCTGTATGGC AGCCAGACCC CGGATGAAGA TTGCCTGTTT


301
CTGGAACGCC TGGAAGAAAA CCATTATAAC ACCTATACCA GCAAAAAACA TGCGGAAAAA


361
AACTGGTTTG TGGGCCTGAA AAAAAACGGC AGCTGCAAAC GCGGCCCGCG CACCCATTAT


421
GGCCAGAAAG CGATTCTGTT TCTGCCGCTG CCGGTGAGCA GCGAT


 







Chinese tree shrew FGF1 gene coding sequence (1-155) (SEQ ID NO: 89)


(generated using SMS Reverse Translate tool on the ExPASy Bioinformatics


Resource website (www.expasy.org):








1
ATGGCGGAAG GCGAAATTAC CACCTTTGCG GCGCTGACCG AAAAATTTGA TCTGCCGCCG


61
GGCAACTATA AAAAACCGAA ACTGCTGTAT TGCAGCAACG GCGGCCATTT TCTGCGCATT


121
CTGCCGGATG GCACCGTGGA TGGCACCCGC GATCGCAGCG ATCAGCATAT TCAGCTGCAG


181
CTGACCGCGG AAAACGTGGG CGAAGTGTAT ATTAAAAGCA CCGAAACCGG CCAGTATCTG


241
GCGATGGATG CGGATGGCCT GCTGTATGGC AGCCAGACCC CGAACGAAGA ATGCCTGTTT


301
CTGGAACGCC TGGAAGAAAA CCATTATAAC ACCTATATTA GCAAAAAACA TGCGGAAAAA


361
AACTGGTTTG TGGCGCTGAA AAAAAACGGC AGCTGCAAAC TGGGCCCGCG CACCCATTAT


421
GGCCAGAAAG CGATTCTGTT TCTGCCGCTG CCGGTGAGCA GCGAT


 







Rock pigeon FGF1 gene coding sequence (1-155) (SEQ ID NO: 90) (generated


using SMS Reverse Translate tool on the ExPASy Bioinformatics Resource


website (www.expasy.org):








1
ATGGCGGAAG GCGAAATTAC CACCTTTACC GCGCTGACCG AAAAATTTAA CCTGCCGCCG


61
GGCAACTATA AAAAACCGAA ACTGCTGTAT TGCAGCAACG GCGGCCATTT TCTGCGCATT


121
CTGCCGGATG GCAAAGTGGA TGGCACCCGC GATCGCAGCG ATCAGCATAT TCAGCTGCAG


181
CTGAGCGCGG AAAGCGTGGG CGAAGTGTAT ATTAAAAGCA CCCAGAGCGG CCAGTATCTG


241
GCGATGGATC CGACCGGCCT GCTGTATGGC AGCCAGCTGC TGGGCGAAGA ATGCCTGTTT


301
CTGGAACGCA TTGAAGAAAA CCATTATAAC ACCTATGTGA GCAAAAAACA TGCGGATAAA


361
AACTGGTTTG TGGGCCTGAA AAAAAACGGC AACAGCAAAC TGGGCCCGCG CACCCATTAT


421
GGCCAGAAAG CGATTCTGTT TCTGCCGCTG CCGGTGAGCG CGGAT


 







Sheep FGF1 gene coding sequence (1-155) (SEQ ID NO: 91)


(GenBank accession no. XM_004008909, which is


hereby incorporated by reference in its entirety):








361
ATGGCTGAAG GAGAAACCAC AACCTTCAGG GCCCTGACTG AGAAGTTTAA CCTGCCTCTA


421
GGCAATTACA AGAAGCCCAA GCTCCTCTAT TGCAGCAACG GGGGCTACTT CCTGAGAATC


481
CTCCCAGATG GCAGAGTGGA TGGGACGAAG GACAGGAGCG ACCAGCACAT TCAGCTGCAG


541
CTCTATGCGG AAAGCATAGG GGAGGTGTAT ATTAAGAGTA CGGAGACTGG CCAGTTCTTG


601
GCCATGGACA CCAACGGGCT TTTGTACGGC TCACAAACAC CCAGTGAGGA ATGTTTGTTC


661
CTGGAAAGGC TGGAGGAAAA CCATTATAAC ACCTACATAT CCAAGAAGCA TGCAGAGAAG


721
AATTGGTTCA TTGGTCTCAA GAAGAACGGA AGCTCCAAAC TCGGTCCTCG GACTCACTTC


781
GGCCAGAAAG CCATCTTGTT TCTCCCCCTG CCAGTTTCCT CTGATTAA


 







Chicken FGF1 gene coding sequence (1-155) (SEQ ID NO: 92) (GenBank


accession no. NM_205180, which is hereby incorporated


by reference in its entirety):








52
                                                        ATGGCCGAG


61
GGGGAGATAA CCACCTTCAC CGCCCTGACC GAGCGCTTCG GCCTGCCGCT GGGCAACTAC


121
AAGAAGCCCA AACTCCTGTA CTGCAGCAAC GGGGGCCACT TCCTACGGAT CCTGCCGGAC


181
GGCAAGGTGG ACGGGACGCG GGACCGGAGT GACCAGCACA TTCAGCTGCA GCTCAGCGCG


241
GAAGATGTGG GCGAGGTCTA TATAAAGAGC ACAGCGTCGG GGCAGTACCT GGCAATGGAC


301
ACCAACGGGC TCCTGTATGG CTCGCAGCTA CCAGGCGAGG AGTGCTTGTT CCTTGAGAGG


361
CTCGAGGAGA ACCATTACAA CACATACATC TCCAAAAAGC ACGCAGACAA GAACTGGTTC


421
GTCGGGCTGA AGAAAAACGG GAACAGCAAG CTGGGGCCGC GGACTCACTA TGGGCAAAAG


481
GCGATCCTCT TCCTCCCATT GCCGGTGTCG GCTGACTGA


 







Alpaca FGF1 gene coding sequence (1-155, excluding 1-57) (SEQ ID NO: 93)


(Ensembl accession no. ENSVPAT00000008395, which is hereby incorporated


by reference in its entirety):








1
CAGCTGCAGC TCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTAC CGAGACTGGC


61
CAGTACTTGG CCATGGACAC CGACGGGCTT TTGCACGGCT CACAGACACC AAATGAGGAA


121
TGTTTGTTCC TGGAAAGGCT GGAGGAGAAC CATTACAACA CCTACACGTC CAAGAAGCAC


181
GCCGAAAAGA ATTGGTTTGT TGGTCTCAAG AAGAATGGAA GCTGCAAACG CGGTCCTCGG


241
ACTCACTACG GCCAGAAGGC GATCTTGTTT CTCCCCTTGC CAGTCTCCTC TGATTAA


 







Anole lizard FGF1 gene coding sequence (1-155) (SEQ ID NO: 94) (Ensembl


accession no. ENSACAT00000013467, which is hereby incorporated by


reference in its entirety):








1
ATGGCTGAAG GTGAAATAAC AACATTCACA GCCTTGACCG AGAGGTTTGC TCTCCCAATG


61
GAGAATTACA AGAAGCCCAA ACTCCTGTAT TGCAGCAATG GAGGCCACTT CCTGAGGATC


121
CTTCCAGATG GAAAAGTGGA TGGCACCATG GACCGGAATG ACAGCTATAT TCAGTTGCTG


181
TTAACAGCAG AAGATGTGGG TGTGGTATAT ATAAAAGGCA CTGAGACCGG GCAGTACTTG


241
GCCATGGATG CCAATGGACA TTTATATGGC TCGCAGTTGC CAACAGAAGA GTGTTTATTT


301
GTGGAAACGC TGGAAGAAAA CCATTACAAT ACATATACCT CAAAGATGCA TGGCGATAAG


361
AAGTGGTATG TTGGCTTGAA AAAGAATGGG AAAGGCAAAC TGGGGCCACG GACTCATCGC


421
GGCCAAAAGG CAATACTTTT CCTTCCACTG CCAGTATCAC CTGATTAG


 







Bushbaby FGF1 gene coding sequence (1-155) (SEQ ID NO: 95) (Ensembl


accession no. ENSOGAT00000005081, which is hereby incorporated by


reference in its entirety):








1
ATGGCTGAAG GGGAAATCAC AACCTTCACA GCCCTCACAG AGAAGTTTAA TCTGCCTCTA


61
GGAAATTACA AGAAGCCCAA GCTCCTCTAC TGTAGCAACG GGGGTCACTT TCTGAGGATC


121
CTGCCGGATG GCACCGTGGA TGGGACACAA GACAGGAGCG ACCAGCACAT TCAGCTGCAG


181
CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CCCAGACTGG CCAGTACTTG


241
GCCATGGACT CCGACGGGCT TTTATACGGC TCACAAACAC CAAATGAGGA ATGCCTGTTC


301
CTGGAACGGC TGGAGGAAAA CCATTACAAC ACCTATGTGT CCAAGAAGCA CGCCGAGAAG


361
AATTGGTTTG TCGGTCTCAA GAAGAACGGA AGTTGCAAAC GTGGTCCTCG GACTCACTAC


421
GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTCTCCT CTGATTAA


 







Cat FGF1 gene coding sequence (1-155) (SEQ ID NO: 96) (Ensembl accession


no. ENSFCAT00000009123, which is hereby incorporated by reference in its


entirety):








1
ATGGCTGAAG GGGAAATCAC AACCTTCACG GCCCTGACGG AGAAGTTCAA TCTGCCTCCA


61
GGGAATTACA AGAAACCCAA ACTCCTCTAC TGTAGCAACG GGGGCCACTT CCTGAGGATC


121
CTTCCAGATG GCACAGTGGA TGGGACGAGG GACAGGAGCG ACCAGCACAT TCAGCTGCAG


181
CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CCGAGACTGG CCAGTACTTG


241
GCCATGGACA CCGACGGGCT TTTGTACGGC TCACAGACAC CAAATGAGGA ATGCTTGTTC


301
CTGGAAAGGC TGGAAGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCA CGCAGAAAAG


361
AATTGGTTTG TGGGTCTCAA GAAGAATGGA AGCTGCAAAC GCGGTCCCCG GACTCACTAT


421
GGCCAGAAGG CAATTTTGTT TCTCCCCCTG CCAGTCTCCT CTGATTAA


 







Chinese softshell turtle FGF1 gene coding sequence (1-155)


(SEQ ID NO: 97) (Ensembl accession no. ENSPSIT00000016432,


which is hereby incorporated by reference in its entirety):








131
           ATGGCTGAAG GGGAAATAAC AACGTTCACC GCCCTGACCG AAAAATTCAA


181
CCTTCCCCTG GGGAATTACA AGAATCCCAA ACTCTTATAT TGCAGCAATG GAGGCTACTT


241
CTTGAGGATA CATCCAGATG GCAAAGTAGA TGGGACAAGG GACCGAAGTG ACCAACACAT


301
TCAGCTGCAG CTAAGTGCGG AAAGCGTGGG TGAGGTATAT ATAAAGAGCA CTGAGTCTGG


361
ACAGTTTTTG GCTATGGACG CCAATGGACT TTTATATGGA TCACTGTCAC CGAGTGAGGA


291
ATGCTTATTC TTGGAAAGAA TGGAAGAAAA TCATTATAAC ACCTACATCT CCAAGAAGCA


351
TGCAGACAAG AACTGGTTCG TTGGCTTAAA GAAGAATGGA AGCTGCAAAC TGGGACCGCG


411
GACGCACTAC GGCCAAAAGG CCGTCCTTTT CCTTCCACTG CCAGTGTCAG CTGATTAA


 







Coelacanth FGF1 gene coding sequence (1-155) (SEQ ID NO: 98) (Ensembl


accession no. ENSLACT00000015212, which is hereby incorporated by


reference in its entirety):








1
ATGGCTGAAG ACAAAATAAC AACACTGAAG GCCTTGGCTG AAAAATTTAA CCTTCCTATG


61
GGAAATTACA AGAAAGCAAA ACTCCTCTAC TGCAGCAACG GAGGGTATTT CCTGCGAATA


121
CCCCCAGACG GGAAAGTGGA AGGAATTAGA GAACGAAGCG ACAAGTACAT TCAGCTGCAA


181
ATGAATGCAG AAAGTTTAGG CATGGTGTCT ATAAAGGGTG TGGAGGCAGG GCAATACCTA


241
GCTATGAATA CAAATGGACT CCTGTATGGA TCTCAGTCTC TAACTGAAGA ATGCCTTTTC


301
ATGGAAAAGA TGGAAGAAAA CCACTACAAC ACATACAGGT CTAAGACACA TGCAGATAAA


361
AACTGGTATG TTGGCATTAG AAAGAACGGT AGCATCAAAC CAGGACCAAG GACTCACATT


421
GGCCAAAAGG CTGTTCTTTT TCTCCCTCTG CCTGCCTCGA GTGATTAG


 







Dolphin FGF1 gene coding sequence (1-155) (SEQ ID NO: 99) (Ensembl


accession no. ENSTTRT00000004742, which is hereby incorporated by


reference in its entirety):








1
ATGGCTGAAG GGGAAATCAC AACCTTCACA GCCCTGACCG AGAAGTTTAA TCTGCCTCCA


61
GGGAATTACA AGAAGCCCAA ACTCCTCTAC TGTAGCAACG GGGGCCACTT CCTGAGGATC


121
CTTCCAGATG GCACAGTGGA TGGGACAAGG GACAGGAGTG ACCAGCACAT TCAGCTGCAG


181
CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CGGAGACTGG CCAGTACTTG


241
GCCATGGACA CCGACGGGCT TTTGTACGGC TCACAGACAC CCAATGAGGA ATGTTTGTTC


301
CTGGAAAGGT TGGAGGAAAA CCATTACAAC ACCTACGCAT CCAAGAAGCA TGCAGAAAAG


361
AATTGGTTCG TTGGTCTCAA GAAGAACGGA AGCTGCAAAC GCGGTCCTCG GACTCACTAC


421
GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTCTCCT CCGATTAA


 







Ferret FGF1 gene coding sequence (1-155) (SEQ ID NO: 100) (Ensembl


accession no. ENSMPUT00000008013, which is hereby incorporated by


reference in its entirety):








1
                                     ATGGCT GAAGGGGAAA TCACAACCTT


61
CACAGCCCTG ATGGAGAAGT TTAATCTGCC TGCGGGGAAT TACAAGAAGC CCAAACTCCT


121
CTACTGTAGC AATGGGGGCC ACTTCCTGAG GATCCTTCCA GATGGCACAG TGGACGGCAC


181
AAGGGACAGG AGCGACCAGC ACATTCAGCT GCAGCTCAGT GCGGAAAGCG TGGGGGAGGT


241
GTACATAAAG AGTACCGAGA CTGGCCAGTA CTTGGCCATG GACACCGATG GGCTTTTGTA


301
CGGCTCACAA ACACCAAATG AGGAATGTCT GTTCCTGGAA AGGCTGGAGG AAAACCATTA


361
CAACACCTAC ACATCCAAGA AGCACGCTGA GAAGAATTGG TTTGTAGGTC TCAAGAAGAA


421
CGGAAGCTGC AAACGCGGTC CTCGGACTCA CTATGGCCAG AAAGCAATTC TGTTTCTCCC


481
CCTGCCAGTC TCCTCTGATT AA


 







Gibbon FGF1 gene coding sequence (1-155) (SEQ ID NO: 101) (Ensembl


accession no. ENSNLET00000012455, which is hereby incorporated by


reference in its entirety):








241
                                                  ATGG CCGAAGGGGA


301
AATCACCACC TTCACAGCCC TGACCGAGAA GTTTAATCTG CCTCCAGGGA ATTACAAGAA


361
GCCCAAACTC CTCTACTGTA GCAACGGGGG CCACTTCTTG AGGATCCTTC CGGATGGCAC


421
AGTGGATGGG ACAAGGGACA GGAGCGACCA GCACATTCAG CTGCAGCTCA GTGCGGAAAG


481
CGTGGGGGAG GTGTATATAA AGAGTACCGA GACTGGCCAG TACTTGGCCA TGGACACCGA


541
CGGGCTTTTA TACGGCTCAC AGACACCAAA TGAGGAATGT TTGTTCCTGG AAAGGCTGGA


601
GGAGAACCAT TACAACACCT ATATATCCAA GAAGCATGCA GAGAAGAATT GGTTTGTTGG


661
CCTCAAGAAG AATGGAAGCT GCAAACGCGG TCCTCGGACT CACTATGGCC AGAAAGCAAT


721
CTTGTTTCTC CCCCTGCCAG TCTCTTCTGA TTAA


 







Gorilla FGF1 gene coding sequence (1-155) (SEQ ID NO: 102) (Ensembl


accession no. ENSGGOT00000025344, which is hereby incorporated by


reference in its entirety):








121
                                                  ATGG CTGAAGGGGA


181
AATCACCACC TTCACAGCCC TGACCGAGAA GTTTAATCTG CCTCCAGGGA ATTACAAGAA


241
GCCCAAACTC CTCTACTGTA GCAATGGGGG CCACTTCTTG AGGATCCTTC CGGATGGCAC


301
AGTGGATGGG ACAAGGGACA GGAGCGACCA GCACATTCAG CTGCAGCTCA GTGCGGAAAG


361
CGTGGGGGAG GTGTATATAA AGAGTACCGA GACTGGCCAG TACTTGGCCA TGGACACCGA


421
CGGGCTTTTA TACGGCTCAC AGACACCAAA TGAGGAATGT TTGTTCCTGG AAAGGCTGGA


481
GGAGAACCAT TACAACACCT ATATATCCAA GAAGCATGCA GAGAAGAATT GGTTTGTTGG


541
CCTCAAGAAG AATGGAAGCT GCAAACGCGG TCCTCGGACT CACTATGGCC AGAAAGCAAT


601
CTTGTTTCTC CCCCTGCCAG TCTCTTCCGA TTAA


 







Hedgehog FGF1 gene coding sequence (1-155) (SEQ ID NO: 103) (Ensembl


accession no. ENSEEUT00000005832, which is hereby incorporated by


reference in its entirety):








1
ATGGCTGAAG GAGAAATCAC CACCTTCACG GCCCTGACTG AGAAGTTTAA TCTGCCACTA


61
GGGAATTACA AGAAGCCCAA GCTCCTCTAC TGTAGCAACG GGGGCCACTT CCTGAGGATC


121
CTTCCAGATG GCACCGTGGA TGGGACAAGG GACAGGAGCG ACCAGCATAT TCAGCTGCAG


181
CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CGGAGACTGG CCAGTACTTG


241
GCCATGGACA CCGACGGGCT TTTATACGGC TCACAAACAC CAAATGAGGA ATGTCTGTTC


301
CTTGAAAGGC TGGAAGAGAA CCATTACAAT ACCTACACAT CCAAGAAGCA TGCCGAGAAG


361
AACTGGTTTG TTGGCCTCAA GAAGAATGGA AGCTGCAAGC GTGGTCCTCG GACTCATTAT


421
GGCCAGAAAG CTATTTTGTT TCTCCCCCTG CCAGTTTCCT CTGATTAA


 







Hyrax FGF1 gene coding sequence (1-155, excluding 1-90) (SEQ ID NO: 104)


(Ensembl accession no. ENSPCAT00000011746, which is hereby incorporated


by reference in its entirety):








1
ATGGCTGAAG GCGAAATCAC AACCTTCACA GCCCTGACTG AGAAGTTTAA CCTGCCACTA


61
GAGAATTACA AGAAGCCCAA ACTCCTCTAC TGTAGCAACG GAGGCCACTT CCTGAGGATC


121
CTTCCGGACG GCACAGTGGA TGGCACCAGG GACAGGAGTG ACCAGCACAT TCAGCTGCAG


181
CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGGGCA CCGAGACTGG CCAGTACTTG


241
GCCATGGACA CCGACGGGCT TTTATATGGC TCA


 







Kangaroo rat FGF1 gene coding sequence (1-155, excluding 1-16 and


58-155) (SEQ ID NO: 105) (Ensembl accession no. ENSDORT00000007345,


which is hereby incorporated by reference in its entirety):








1
ATGGCTGAAG GGGAAATCAC AACCTTCACA GCCCTGACGG AAAGGTTTAA ----------



---------- ---------- ---------- ---------- ---------- ----------


51
---------- ---------- ---------- ---------- ---------T TCAGCTGCAA


62
CTGAGTGCGG AAAGCGTGGG GGAGGTCTAT ATAAAGAGCA CCGAGACTGG CCAATACTTG


122
GCCATGGATG CCGACGGGCT TTTATACGGC TCACAGACAC CTGATGAAGA ATGCTTGTTC


182
CTGGAGAGGC TGGAAGAAAA TCATTATAAC ACCTACATAG CCAAGAAACA TGCTGAAAAG


242
AATTGGTTTG TCGGCCTCAA AAAGAATGGA AGCTGCAAGC GTGGTCCTCG GACTCACTAT


302
GGCCAGAAAG CAATCCTGTT CCTCCCCTTG CCTGTCTCCT CTGATTAG


 







Lamprey FGF1 gene coding sequence (1-155, excluding 94-155) (SEQ ID


NO: 106) (Ensembl accession no. ENSPMAT00000010729, which is hereby


incorporated by reference in its entirety):








1
ATGGAGGTGG GCCACATCGG CACGCTGCCC GTGGTCCCCG CGGGGCCCGT GTTCCCCGGC


61
AGTTTCAAGG AGCCACGGCG CCTCTACTGC CGCAGCGCGG GCCACCACCT CCAGATCCTG


121
GGGGACGGCA CCGTGAGTGG CACCCAGGAC GAGAACGAGC CCCACGCCGT TCTGCAGCTG


181
CAGGCGGTGC GCCGCGGGGT GGTGACGATC CGTGGGCTCT GCGCCGAGAG GTTCCTCGCC


241
ATGAGCACGG AGGGACACCT GTACGGGGCG GTGAGG


 







Lesser hedgehog tenrec FGF1 gene coding sequence (1-155, excluding 1-57)


(SEQ ID NO: 107) (Ensembl accession no. ENSETET00000017851, which is


hereby incorporated by reference in its entirety):








1
CAGCTGAAGC TCGTTGCCGA AAGCGTGGGG GTGGTGTATA TAAAGAGCAT CAAGACCGGC


61
CAGTACTTGG CCATGAACCC CGACGGGCTT TTATACGGCT CCGAGACCCC AGAGGAAGAA


121
TGCTTGTTCC TGGAAACGCT GGAGGAAAAC CACTACACCA CCTTCAAATC TAAGAAGCAC


181
GTAGAGAAGA ATTGGTTCGT TGGTCTCCGG AAGAATGGAA GGGTCAAGAT CGGGCCTCGG


241
ACTCACCAAG GCCAGAAAGC AATCTTGTTC CTGCCCCTCC CGGTGTCCTC TGATTAA


 







Rhesus monkey FGF1 gene coding sequence (1-155) (SEQ ID NO: 108)


(Ensembl accession no. ENSMMUT00000033070, which is hereby


incorporated by reference in its entirety):








36
                                      ATGGC TGAAGGGGAA ATCACCACGT


61
TCACAGCCCT GACCGAGAAG TTTAATCTGC CTCCAGGGAA TTACAAGAAG CCCAAACTGC


121
TCTACTGTAG CAATGGGGGC CACTTCTTGA GGATCCTTCC GGATGGCACA GTGGATGGGA


181
CAAGGGACAG GAGCGACCAG CACATTCAGC TGCAGCTCAG TGCGGAAAGC GTGGGGGAGG


241
TGTATATAAA GAGTACCGAG ACTGGCCAGT ACTTGGCCAT GGACACCGAC GGGCTTTTAT


301
ACGGCTCACA GACACCAAAT GAGGAATGTT TGTTCCTGGA AAGGCTGGAG GAGAACCATT


361
ACAACACCTA TACATCCAAG AAGCACGCAG AGAAGAATTG GTTTGTTGGC CTCAAGAAGA


421
ATGGAAGCTG CAAACGTGGT CCTCGGACTC ACTATGGCCA GAAAGCAATC TTGTTTCTTC


481
CCCTGCCAGT CTCTTCTGAT TAA


 







Megabat FGF1 gene coding sequence (1-155) (SEQ ID NO: 109) (Ensembl


accession no. ENSPVAT00000004596, which is hereby incorporated by


reference in its entirety):








1
ATGGCCGAGG GGGAAGTCAC GACGTTCACG GCCCTGACCG AGAGGTTTAA CCTGCCTCCA


61
GGGAATTACA AGAAGCCCAA ACTTCTCTAC TGCAGCAACG GGGGCCACTT CCTGAGGATC


121
CTCCCAGATG GCACAGTGGA TGGGACAAGG GACAAGAGCG ACCAGCACAT TCAGCTGCAG


181
CTCAGTGCGG AAAGTGTGGG GGAGGTGTAT ATAAAGAGCA CCGAGAGTGG CCAGTACTTG


241
GCCATGGACT CCGACGGGCT TTTGTACGGC TCACAGACAC CAGATGAGGA CTGTTTGTTC


301
CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCA CGCAGAGAAG


361
AATTGGTTTG TTGGGCTCAA GAAGAATGGA AGCTGCAAGC GCGGTCCCCG GACTCACTAC


421
GGCCAGAAAG CGATCCTGTT TCTCCCCCTG CCAGTCTCCT CTGATTAG


 







Microbat FGF1 gene coding sequence (1-155) (SEQ ID NO: 110) (Ensembl


accession no. ENSMLUT00000007098, which is hereby incorporated by


reference in its entirety):








66
     ATGGC TGAGGGGGAA GTCACCACAT TCACGGCCCT GACCGAGAGG TTCAATCTGC


121
CTCTGGAGAA CTACAAGAAG CCCAAGCTTC TCTACTGCAG CAACGGGGGC CACTTCCTGC


181
GGATCCTCCC AGACGGCACC GTGGACGGGA CGAGGGACAG GAGCGACCAG CACATTCAGC


241
TGCAGCTCAG TGCGGAAAGC GTGGGGGAGG TGTATATAAA GAGCACCGAG AGTGGCCAGT


301
ACTTGGCCAT GGACTCCGAC GGGCTTTTGT ACGGCTCACA AACACCCAAT GAGGAATGTT


361
TGTTCCTGGA AAGGCTGGAG GAGAACCACT ACAACACCTA CACGTCCAAG AAGCACGCAG


421
AAAAGAATTG GTTCGTTGGG CTCAAGAAGA ACGGAAGCTG CAAGCGTGGT CCTCGGACGC


481
ATTATGGCCA GAAAGCAATC TTGTTTCTCC CCCTGCCAGT CTCCTCCGAT TAA


 







Mouse lemur FGF1 gene coding sequence (1-155) (SEQ ID NO: 111) (Ensembl


accession no. ENSMICT00000009454, which is hereby incorporated by


reference in its entirety):








1
ATGGCCGAAG GGGAGATCAC AACCTTCACG GCCCTCACCG AGAAGTTTAA CCTGCCTCCG


61
GGGAACTACA AGAAGCCCAA GCTCCTCTAC TGCAGCAACG GCGGCCACTT CCTGCGCATC


121
CTTCCCGACG GCACCGTGGA TGGCACGAGA GACAGGAGCG ACCAGCACAT TCAGCTGCAG


181
CTCAGTGCGG AAAGCGCGGG GGAGGTGTAT ATAAAGAGCA CCCAGACTGG CCGGTACTTG


241
GCCATGGACG CCGACGGGCT TTTATACGGC TCACAAACAC CAAATGAGGA ATGTTTGTTC


301
CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACGTAT CCAAGAAGCA CGCAGAGAAG


361
AATTGGTTTG TTGGCCTCAA GAAGAATGGA AGTTGCAAAC GCGGCCCCCG GACTCACTAT


421
GGCCAGAAAG CAATCTTGTT TCTGCCCCTG CCAGTCTCCT CTGATTAA


 







Pika FGF1 gene coding sequence (1-155, excluding 57-67) (SEQ ID NO: 112)


(Ensembl accession no. ENSOPRT00000012854, which is hereby incorporated


by reference in its entirety):








1
ATGGCCGAGG GAGAAGTCAC CACCTTCTCA GCCCTGACGG AGAAGTTCAA TCTGCCTGGA


61
GGAAACTACA AGTTGCCCAA GCTCCTTTAC TGTAGCAACG GAGGCCACTT CCTGAGGATC


121
CTTCCAGATG GCACAGTGGA TGGGACCAGG GACAGGAGCG ACCTGCACA- ----------


170
---------- ---------- -GAGGTGTTT ATAAAGAGTA CGGAGACTGG CCAGTACTTG


209
GCTATGGACA CCGATGGCCT TTTATATGGC TCGCAGACAC CCAGTGAGGA GTGTTTGTTC


269
CTGGAGCGGC TGGAGGAGAA CCACTACAAC ACCTACACAT CCAAGAAGCA TGCCGAGAAG


329
AACTGGTTTG TGGGCATCAA GAAGAATGGA AGCTGCAAGC GTGGTCCTCG GACTCACTAC


389
GGCCAGAAAG CCATCTTGTT TCTCCCTCTG CCAGTCTCTT CTGACTAA


 







Rat FGF1 gene coding sequence (1-155) (SEQ ID NO: 113)


(Ensembl accession no. ENSRNOT00000018577, which is


hereby incorporated by reference in its entirety):








268
                             ATG GCCGAAGGGG AGATCACAAC CTTTGCAGCC


301
CTGACCGAGA GGTTCAATCT GCCTCTAGGG AACTACAAAA AACCCAAACT GCTCTACTGC


361
AGCAACGGGG GCCACTTCTT GAGGATTCTT CCCGATGGCA CCGTGGATGG GACCAGGGAC


421
AGGAGCGACC AGCACATTCA GCTGCAGCTC AGTGCGGAAA GCGCGGGCGA AGTGTATATA


481
AAGGGTACAG AGACTGGCCA GTACTTGGCC ATGGACACCG AAGGGCTTTT ATACGGCTCG


541
CAGACACCAA ATGAAGAATG CCTATTCCTG GAAAGGCTAG AAGAAAACCA TTATAACACT


601
TACACATCCA AGAAGCACGC GGAGAAGAAC TGGTTTGTGG GCCTCAAGAA GAACGGGAGT


661
TGTAAGCGCG GTCCTCGGAC TCACTACGGC CAGAAAGCCA TCTTGTTTCT CCCCCTCCCG


721
GTATCTTCTG ACTAA


 







Sloth FGF1 gene coding sequence (1-155) (SEQ ID NO: 114) (Ensembl


accession no. ENSCHOT00000012416, which is hereby incorporated by


reference in its entirety):








1
ATGGCTGAAG GGGAAATCAC AACCTTCACA GCTCTGATGG AGAAGTTTAA CCTGCCACCA


61
GGGAATTACA TGAAGCCCAA ACTCCTCTAC TGTAGCAACG GGGGCCACTT CTTGAGGATC


121
CTTCCAGACG GCACAGTGGA TGGGACAAGG GACAGGAGCG ACCTGCACAT TCAGCTGCAG


181
CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTG CGGAGACCGG CCAGTACTTA


241
GCCATGGACA CCGGCGGGCT TTTATACGGC TCACAGACAC CAAGTGAGGA ATGCCTGTTC


301
CTAGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACGTAT CCAAGAAGCA TGCGGAGAAG


361
AACTGGTTCG TTGGCCTAAA GAAGAATGGA AGCAGCAAAC GCGGCCCCCG GACTCACTAT


421
GGCCAGAAAG CCATCTTGTT TCTTCCCCTG CCAGTCTCCT CTGATTAA


 







Squirrel FGF1 gene coding sequence (1-155) (SEQ ID NO: 115) (Ensembl


accession no. ENSSTOT00000029249, which is hereby incorporated by


reference in its entirety):








1
                                                             ATGG


5
CTGAAGGGGA AATCACAACC TTCACAGCCC TGACCGAGAA GTTCAATCTG CCTCCAGGGA


65
ACTACAAGAA GCCCAAACTG CTCTACTGTA GCAACGGAGG CCACTTCTTG AGGATCCTTC


125
CTGATGGCAC AGTGGATGGG ACAAGAGACA GGAGCGACCA ACACATTCAG CTGCAGCTCA


185
GTGCGGAAAG CGTGGGGGAG GTGTATATAA AGAGTACCGA GACCGGCCAG TACTTGGCCA


245
TGGACACCGA CGGGCTTTTA TATGGCTCAC AGACCCCAAA TGAGGAATGC TTATTCCTGG


305
AAAGGCTGGA GGAAAACCAT TACAACACGT ACACATCCAA GAAGCATGCA GAGAAGAATT


365
GGTTTGTTGG CCTCAAGAAG AACGGAAGCT GCAAGCGCGG TCCCCGGACT CACTATGGCC


425
AGAAAGCGAT CTTGTTTCTC CCACTGCCTG TCTCCTCTGA TTAG


 







Tarsier FGF1 gene coding sequence (1-155) (SEQ ID NO: 116)


(Ensembl accession no. ENSTSYT00000007425, which is hereby


incorporated by reference in its entirety):








1
ATGGCCGAAG GGGAAATCAC AACCTTCACA GCCCTGACCG AGAAGTTCAA CCTGCCCCCG


61
GGGAATTACA AGAAGCCCAA ACTCCTCTAC TGCAGCAACG GGGGCCACTT CTTGAGGATC


121
CTTCCGGATG GCACTGTGGA TGGAACGAGG GACAGGAGCG ACCAGCACAT TCAGCTGCAG


181
CTCAGCGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CCGAGACCGG CCAGTACTTG


241
GCCATGGACA CCGACGGGCT TTTGTACGGC TCACAGACAC CAAATGAGGA GTGTCTGTTC


301
CTGGAAAGGC TGGAAGAGAA TCATTACAAT ACCTACGTGT CCAAGAAGCA TGCGGAGAAG


361
AATTGGTTTG TCGGCCTCAA GAAGAATGGA AGCTGCAAAC GCGGTCCTCG GACTCACTAT


421
GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTTTCCT CTGATTAA


 







Tree shrew FGF1 gene coding sequence (1-155) (SEQ ID NO: 117)


(Ensembl accession no. ENSTBET00000011861, which is hereby


incorporated by reference in its entirety):








1
ATGGCTGAAG GGGAAATCAC GACCTTCGCA GCCCTGACCG AGAAGTTTGA TCTGCCTCCA


61
GGGAATTACA AGAAGCCCAA ACTTCTCTAC TGTAGCAACG GGGGCCATTT CTTGAGGATT


121
CTTCCAGATG GCACCGTGGA TGGGACAAGA GACAGGAGCG ACCAGCACAT TCAGCTGCAG


181
CTCACTGCGG AAAACGTGGG GGAGGTGTAC ATAAAGAGTA CGGAGACTGG CCAGTACTTG


241
GCCATGGACG CCGACGGGCT TTTATATGGC TCACAGACAC CAAACGAGGA ATGTTTGTTC


301
CTGGAAAGGC TGGAGGAGAA CCATTACAAC ACCTACATAT CCAAGAAGCA CGCAGAGAAG


361
AATTGGTTTG TTGCCCTCAA GAAGAACGGA AGCTGCAAAC TCGGTCCTCG GACTCACTAT


421
GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTCTCCT CTGATTAA


 







Turkey FGF1 gene coding sequence (1-155, excluding 57-155)


(SEQ ID NO: 118) (Ensembl accession no. ENSMGAT00000017372,


which is hereby incorporated by reference in its entirety):








1
ATGGCCGAGG GGGAGATAAC CACCTTCACA GCCCTGACCG AGCGCTTCGG CCTGCCGCTG


61
GGCAACTACA AGAAGCCCAA ACTCCTGTAC TGCAGCAACG GGGGCCACTT CCTACGGATC


121
CTGCCGGACG GCAAGGTGGA CGGGACGCGG GACCGGAGCG ACCAGCAC


 







Wallaby FGF1 gene coding sequence (1-155) (SEQ ID NO: 119)


(Ensembl accession no. ENSMEUT00000016544, which is hereby


incorporated by reference in its entirety):








1
ATGGCCGAAG GGGAGATCAC AACCTTCACA GCCCTGACCG AAAGATTTAA CCTGCCACTG


61
GGGAATTACA AGAAGCCCAA GCTTCTCTAC TGTAGCAATG GGGGCCACTT TTTGAGGATC


121
CTTCCTGATG GCAAAGTGGA TGGGACAAGG GACAGAAATG ATCAACACAT TCAACTGCAA


181
CTAAGCGCGG AAAGCGTGGG TGAGGTGTAT ATAAAGAGCA CTGAGTCTGG GCAGTATTTG


241
GCCATGGACA CCAATGGACT TTTATATGGC TCACAGACCC CCAGCGAAGA ATGCTTATTC


301
CTGGAGAGGT TGGAGGAGAA TCATTACAAC ACCTACATAT CAAAGAAGCA TGCGGAGAAA


361
AATTGGTTTG TTGGCCTCAA GAAGAACGGA AGTTGCAAAA GAGGTCCCAG GACTCACTAT


421
GGCCAGAAAG CCATCCTATT CCTTCCCCTC CCTGTGTCCT CTGAGTAA


 







Zebrafish FGF1 gene coding sequence (1-147) (SEQ ID NO: 120)


(Ensembl accession no. ENSDART00000005842, which is hereby


incorporated by reference in its entirety):








178
                                                              ATG


181
ACCGAGGCCG ATATTGCGGT AAAGTCCAGC CCGCGCGACT ATAAAAAACT GACGCGGCTG


241
TACTGTATGA ATGGAGGATT TCACCTTCAG ATCCTGGCGG ACGGGACAGT GGCTGGAGCA


124
GCAGACGAAA ACACATACAG CATACTGCGC ATAAAAGCAA CAAGTCCAGG AGTGGTGGTG


184
ATCGAAGGAT CAGAAACAGG TCTTTACCTC TCGATGAATG AACATGGCAA GCTGTACGCT


244
TCATCATTAG TGACGGATGA AAGTTATTTC CTGGAGAAGA TGGAGGAAAA CCACTACAAC


304
ACATATCAGT CTCAAAAGCA CGGTGAAAAC TGGTACGTCG GAATAAAAAA GAACGGGAAA


364
ATGAAACGGG GCCCAAGAAC TCACATCGGA CAAAAGGCCA TTTTCTTTCT TCCACGACAG


424
GTGGAGCAGG AAGAGGACTG A









As noted above, also encompassed within the present invention are portions of paracrine FGFs other than FGF1 (e.g., FGF2, FGF4, FGF5, FGF6, FGF9, FGF16, and FGF20). The portions derived from paracrine FGF2 include portions corresponding to the above-identified amino acid sequences of FGF1. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
In one embodiment, the paracrine FGF is FGF2. In one embodiment, the portion of the FGF2 is derived from human FGF2 having the amino acid sequence of SEQ ID NO: 121 (GenBank Accession No. EAX05222, which is hereby incorporated by reference in its entirety), as follows:









1
MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI



 


61
KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY


 


121
TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS






In one embodiment, the portion of the paracrine FGF includes an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 151 to 155 of SEQ ID NO: 121. In one embodiment, the portion of the paracrine FGF includes amino acid residues 1-151, 1-152, 1-153, 1-154, 1-155, 2-151, 2-152, 2-153, 2-154, 2-155, 3-151, 3-152, 3-153, 3-154, 3-155, 4-151, 4-152, 4-153, 4-154, 4-155, 5-151, 5-152, 5-153, 5-154, 5-155, 6-151, 6-152, 6-153, 6-154, 6-155, 7-151, 7-152, 7-153, 7-154, 7-155, 8-151, 8-152, 8-153, 8-154, 8-155, 9-151, 9-152, 9-153, 9-154, 9-155, 10-151, 10-152, 10-153, 10-154, 10-155, 11-151, 11-152, 11-153, 11-154, 11-155, 12-151, 12-152, 12-153, 12-154, 12-155, 13-151, 13-152, 13-153, 13-154, 13-155, 14-151, 14-152, 14-153, 14-154, 14-155, 15-151, 15-152, 15-153, 15-154, 15-155, 16-151, 16-152, 16-153, 16-154, 16-155, 17-151, 17-152, 17-153, 17-154, 17-155, 18-151, 18-152, 18-153, 18-154, 18-155, 19-151, 19-152, 19-153, 19-154, 19-155, 20-151, 20-152, 20-153, 20-154, 21-155, 21-151, 21-152, 21-153, 21-154, 21-155, 22-151, 22-152, 22-153, 22-154, 22-155, 23-151, 23-152, 23-153, 23-154, 23-155, 24-151, 24-152, 24-153, 24-154, 24-155, 25-151, 25-152, 25-153, 25-154, or 25-155 of FGF2 (SEQ ID NO: 121). In one embodiment, the portion of the paracrine FGF includes amino acid residues 1-151 or 1-152 of SEQ ID NO: 121.
In one embodiment, the portion of the paracrine FGF of the chimeric protein includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the corresponding amino acid sequence of native paracrine FGF (e.g., SEQ ID NO: 121). In one embodiment, the portion of the paracrine FGF includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 151 to 155 of SEQ ID NO: 121. In one embodiment, the portion of the paracrine FGF includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence homology to the corresponding amino acid sequence of native paracrine FGF (e.g., SEQ ID NO: 121). In one embodiment, the portion of the paracrine FGF includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence homology to an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 151 to 155 of SEQ ID NO: 121.
Also encompassed within the present invention are portions of paracrine FGFs other than FGF2 (e.g., FGF1, FGF4, FGF5, FGF6, FGF9, FGF16, and FGF20). The portions derived from paracrine FGFs other than FGF2 include portions corresponding to the above-identified amino acid sequences of FGF2. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
In one embodiment of the present invention, the portion of the paracrine FGF is derived from an ortholog of a human paracrine FGF. In one embodiment of the present invention, the portion of the paracrine FGF of the chimeric protein is derived from an ortholog of human FGF2. In one embodiment, the portion of the FGF2 is derived from Gorilla gorilla, Pongo abelii, Macaca mulatta, Pan troglodytes, Pan paniscus, Saimiri boliviensis boliviensis, Nomascus leucogenys, Equus caballus, Bos taurus, Papio Anubis, Vicugna pacos, Ovis aries, Capreolus capreolus, Loxodonta Africana, Sus scrofa, Ailuropoda melanoleuca, Choloepus hoffmanni, Bubalus bubalis, Canis lupus familiaris, Rattus norvegicus, Heterocephalus glaber, Otolemur garnettii, Mus musculus, Ictidomys tridecemlineatus, Felis catus, Cavia porcellus, Sarcophilus harrisii, Monodelphis domestica, Oryctolagus cuniculus, Meleagris gallopavo, Gallus gallus, Taeniopygia guttata, Cynops pyrrhogaster, Xenopus laevis, Didelphis albiventris, Myotis lucifugus, Anolis carolinensis, Dasypus novemcinctus, Tupaia belangeri, Xenopus silurana tropicalis, Latimeria chalumnae, Tetraodon nigroviridis, Gasterosteus aculeatus, Takifugu rubripes, Oncorhynchus mykiss, Salmo salar, Danio rerio, Oreochromis niloticus, or Oryzias latipes. The portions of an ortholog of human paracrine FGF include portions corresponding to the above-identified amino acid sequences of FGF2. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.
In one embodiment, the portion of the FGF2 of the chimeric protein of the present invention is derived from an ortholog of human FGF2 having the amino acid sequence shown in Table 3.







TABLE 3







Amino acid sequence of Gorilla gorilla (gorilla) FGF2


(SEQ ID NO: 122) (Ensembl accession no. ENSGGOP00000004720,


which is hereby incorporated by reference in its entirety):








104
                                                MAAGSI TTLPALPEDG


120
GSGAFPPGHF KDPKRLYCKN GGFFLRIHPD GRVDGVREKS DPHIKLQLQA EERGVVSIKG


180
VCANRYLAMK EDGRLLASKC VTDECFFFER LESNNYNTYR SRKYTSWYVA LKRTGQYKLG


240
SKTGPGQKAI LFLPMSAKS


 







Amino acid sequence of Pongo abelii (sumatran orangutan) FGF2(SEQ ID


NO: 123) (GenBank accession no. XP_002815172, which is hereby


incorporated by reference in its entirety):








168
                                                   MAA GSITTLPALP


181
EDGGSGAFPP GHFKDPKRLY CKNGGFFLRI HPDGRVDGVR EKSDPHIKLQ LQAEERGVVS


241
IKGVCANRYL AMKEDGRLLA SKCVTDECFF FERLESNNYN TYRSRKYTSW YVALKRTGQY


301
KLGSKTGPGQ KAILFLPMSA KS


 







Amino acid sequence of Macaca mulatta (rhesus monkey) FGF2 (SEQ ID


NO: 124) (GenBank accession no. XP_001099284, which is hereby


incorporated by reference in its entirety):








83
                        MAAGSITT LPALPEDGGS GAFPPGHFKD PKRLYCKNGG


121
FFLRIHPDGR VDGVREKSDP HIKLQLQAEE RGVVSIKGVC ANRYLAMKED GRLLASKCVT


181
DECFFFERLE SNNYNTYRSR KYTSWYVALK RTGQYKLGSK TGPGQKAILF LPMSAKS


 







Amino acid sequence of Pan troglodytes (chimpanzee) FGF2 (SEQ ID


NO: 125) (GenBank accession no. NP_001103711, which is hereby


incorporated by reference in its entirety):








134
              MAAGSIT TLPALPEDGG SGAFPPGHFK DPKRLYCKNG GFFLRIHPDG


181
RVDGVREKSD PHIKLQLQAE ERGVVSIKGV CANRYLAMKE DGRLLASKCV TDECFFFERL


241
ESNNYNTYRS RKYTSWYVAL KRTGQYKLGS KTGPGQKAIL FLPMSAKS


 







Amino acid sequence of Pan paniscus (Pygmy chimpanzee) FGF2 (SEQ ID


NO: 126) (GenBank accession no. XP_003816481, which is hereby


incorporated by reference in its entirety):








112
                                                        MAAGSITTL


121
PALPEDGGSG AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGVREKSDPH IKLQLQAEER


181
GVVSIKGVCA NRYLAMKEDG RLLASKCVTD ECFFFERLES NNYNTYRSRK YTSWYVALKR


241
TGQYKLGSKT GPGQKAILFL PMSAKS


 







Amino acid sequence of Saimiri boliviensis boliviensis (Bolivian


squirrel monkey) FGF2 (SEQ ID NO: 127) (GenBank accession no.


XP_003936290, which is hereby incorporated by reference in its


entirety):








1
MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI


61
KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY


121
TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS


 







Amino acid sequence of Nomascus leucogenys (Northern white-cheeked


gibbon) FGF2 (SEQ ID NO: 128) (GenBank accession no. XP_003271404,


which is hereby incorporated by reference in its entirety):








1
MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI


61
KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY


121
TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS


 







Amino acid sequence of Equus caballus (horse) FGF2 (SEQ ID NO: 129)


(GenBank accession no. NP_001182150, which is hereby incorporated by


reference in its entirety):








1
MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI


61
KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY


121
SSWYVALKRT GQYKLGPKTG PGQKAILFLP MSAKS


 







Amino acid sequence of Bos taurus (cattle) FGF2 (SEQ ID NO: 130)


(GenBank accession no. NP_776481, which is hereby incorporated by


reference in its entirety):








1
MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI


61
KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY


121
SSWYVALKRT GQYKLGPKTG PGQKAILFLP MASKS


 







Amino acid sequence of Papio anubis (Olive baboon) FGF2 (SEQ ID


NO: 131) (GenBank accession no. XP_003899210, which is hereby


incorporated by reference in its entirety):








1
MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI


61
KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY


121
TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS


 







Amino acid sequence of Vicugna pacos (alpaca) FGF2 (SEQ ID NO: 132)


(Ensembl accession no. ENSVPAP00000009804, which is hereby


incorporated by reference in its entirety):








111
                                                       MAAGSITTLP


121
ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI KLQLQAEERG


181
VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY SSWYVALKRT


241
GQYKLGPKTG PGQKAILFLP MSAKS


 







Amino acid sequence of Ovis aries (sheep) FGF2 (SEQ ID NO: 133)


(GenBank accession no. NP_001009769, which is hereby incorporated by


reference in its entirety):








1
MAAGSITTLP ALPEDGGSSA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI


61
KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY


121
SSWYVALKRT GQYKLGPKTG PGQKAILFLP MSAKS


 







Amino acid sequence of Capreolus capreolus (Western roe deer) FGF2


(partial amino acid sequence corresponding to human FGF2 residues 42


to 149)(SEQ ID NO: 134) (GenBank accession no. AAF73226, which is


hereby incorporated by reference in its entirety):








1
RIHPDGRVDG VREKSDPHIK LQLQAEERGV VSIKGVCANR YLAMKEDGRL LASKCVTDEC


61
FFFERLESNN YNTYRSRKYS SWYVALKRTG QYKLGPKTGP GQKAILFL


 







Amino acid sequence of Loxodonta africana (elephant) FGF2 (partial


amino acid sequence corresponding to human FGF2 residues 60 to 155)


(SEQ ID NO: 135) (Ensembl accession no. ENSLAFP00000008249, which is


hereby incorporated by reference in its entirety):








1
VKLQLQAEER GVVSIKGVCA NRYLAMKEDG RLLASRCVTD ECFFFERLES NNYNTYRSRK


61
YTSWYVALKR TGQYKLGSKT GPGQKAILFL PMSAKS


 







Amino acid sequence of Sus scrofa (pig) FGF2 (partial amino acid


sequence corresponding to human FGF2 residues 36 to 155) (SEQ ID


NO: 136) (GenBank accession no. CAE11791 and Ensembl accession no.


ENSSSCP00000009695, which is hereby incorporated by reference in its


entirety):








1
NGGFFLRIHP DGRVDGVREK SDPHIKLQLQ AEERGVVSIK GVCANRYLAM KEDGRLLASK


61
CVTDECFFFE RLESNNYNTY RSRKYSSWYV ALKRTGQYKL GPKTGPGQKA ILFLPMSAKS


 







Amino acid sequence of Ailuropoda melanoleuca (panda) FGF2 (partial


amino acid sequence corresponding to human FGF2 residues 60 to 155)


(SEQ ID NO: 137) (Ensembl accession no. ENSAMEP00000018489, which


is hereby incorporated by reference in its entirety):








1
VKLQLQAEER GVVSIKGVCA NRYLAMKEDG RLLASKCVTD ECFFFERLES NNYNTYRSRK


61
YSSWYVALKR TGQYKLGPKT GPGQKAILFL PMSAKS


 







Amino acid sequence of Choloepus hoffmanni (sloth) FGF2 (SEQ ID


NO: 138) (Ensembl accession no. ENSCHOP00000010051, which is hereby


incorporated by reference in its entirety):








14
                                                          MAAGSIT


21
TLPALPEDGG SGALPPGHFK DPKRLYCKNG GFFLRIHPDG RVDGVREKSD PHIKLQLQAE


81
ERGVVSIKGV CANRYLAMKE DGRLQASKCV TDECFFFERL ESNNYNTYRS RKYSSWYVAL


141
KRTGQYKLGP KTGPGQKAIL FLPMSAKS


 







Amino acid sequence of Bubalus bubalis (water buffalo) FGF2 (SEQ ID


NO: 139) (GenBank accession no. AFH66795, which is hereby


incorporated by reference in its entirety):








1
MAAGSITTLP PLPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI


61
KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESS NYNTYRSRKY


121
SSWYVALKRT GQYKLGPKTG PGQKAILFLP MSAKS


 







Amino acid sequence of Canis lupus familiaris (dog) FGF2 (SEQ ID


NO: 140) (GenBank accession no. XP_003432529, which is hereby


incorporated by reference in its entirety):








40
                                          M AAGSITTLPA LPEDGGSGAF


61
PPGHFKDPKR LYCKKGGFFL RIHPDGRVDG VREKSDPHVK LQLQAEERGV VSIKGVCANR


121
YLAMKEDGRL LASKCVTDEC FFFERLESNN YNTYRSRKYS SWYVALKRTG QYKLGPKTGP


181
GQKAILFLPM SAKS


 







Amino acid sequence of Rattus norvegicus (Norway rat) FGF2 (SEQ ID


NO: 141) (GenBank accession no. NP_062178, which is hereby


incorporated by reference in its entirety):








1
MAAGSITSLP ALPEDGGGAF PPGHFKDPKR LYCKNGGFFL RIHPDGRVDG VREKSDPHVK


61
LQLQAEERGV VSIKGVCANR YLAMKEDGRL LASKCVTEEC FFFERLESNN YNTYRSRKYS


121
SWYVALKRTG QYKLGSKTGP GQKAILFLPM SAKS


 







Amino acid sequence of Heterocephalus glaber (naked mole-rat) FGF2


(partial amino acid sequence corresponding to human FGF2 residues 22


to 155) (SEQ ID NO: 142) (GenBank accession no. EHB17407, which is


hereby incorporated by reference in its entirety):








1
ppghfkdpkr lycknggffl rihpdgrvdg vreksdphvk lqlqaeergv vsikgvcanr


61
ylamkedgrl laskcvtdec ffferlesnn yntyrsrkys swyvalkrtg qyklgsktgp


121
gqkailflpm saks


 







Amino acid sequence of Otolemur garnettii (bushbaby) FGF2(SEQ ID


NO: 143) (Ensembl accession no. ENSOGAP00000021960, which is hereby


incorporated by reference in its entirety):








52
                                                        MAAGSITTL


61
PSLPEDGGSD AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGVREKSDPY IKLQLQAEER


121
GVVSIKGVCA NRYLAMKEDG RLLASKLITD ECFFFERLES NNYNTYRSRK YSSWYVALKR


181
TGQYKLGSKT GPGQKAILFL PMSAKS


 







Amino acid sequence of Mus musculus (house mouse) FGF2 (SEQ ID


NO: 144) (GenBank accession no. NP_032032, which is


hereby incorporated by reference in its entirety):








1
MAASGITSLP ALPEDGGAAF PPGHFKDPKR LYCKNGGFFL RIHPDGRVDG VREKSDPHVK


61
LQLQAEERGV VSIKGVCANR YLAMKEDGRL LASKCVTEEC FFFERLESNN YNTYRSRKYS


121
SWYVALKRTG QYKLGSKTGP GQKAILFLPM SAKS


 







Amino acid sequence of Ictidomys tridecemlineatus (squirrel) FGF2


(partial amino acid sequence corresponding to human FGF2 residues 12


to 155) (SEQ ID NO: 145) (Ensembl accession no. ENSSTOP00000015653,


which is hereby incorporated by reference in its entirety):








1
LPEDGGGGAF PPGHFKDPKR LYCKNGGFFL RIHPDGRVDG VREKSDPHIK LQLQAEDRGV


61
VSIKGVCANR YLAMKEDGRL LASKCVTDEC FFFERLESNN YNTYRSRKYS SWYVALKRTG


121
QYKLGSKTGP GQKAILFLPM SAKS


 







Amino acid sequence of Felis catus (domestic cat) FGF2 (partial


amino acid sequence corresponding to human FGF2 residues 25 to 130)


(SEQ ID NO: 146) (GenBank accession no. ABY47638, which is


hereby incorporated by reference in its entirety):








1
HFKDPKRLYC KNGGFFLRIH PDGRVDGVRE KSDPHIKLQL QAEERGVVSI KGVCANRYLA


61
MKEDGRLLAS KCVTDECFFF ERLESNNYNT YRSRKYSSWY VALKRT


 







Amino acid sequence of Cavia porcellus (guinea pig) FGF2 (partial


amino acid sequence corresponding to human FGF2 residues 60 to 155)


(SEQ ID NO: 147) (Ensembl accession no. ENSCPOP00000004847, which is


hereby incorporated by reference in its entirety):








1
VKLQLQAEDR GVVSIKGVCA NRYLAMKEDG RLLASKCVTD ECFFFERLES NNYNTYRSRK


61
YSSWYVALKR TGQYKLGSKT GPGQKAILFL PMSAKS


 







Amino acid sequence of Sarcophilus harrisii (Tasmanian devil) FGF2


(SEQ ID NO: 148) (Ensembl accession no. ENSSHAP00000012215, which is


hereby incorporated by reference in its entirety):








48
                                                   MAA GSITTLPALA


61
GDGASGGAFP PGHFQDPKRL YCKNGGFFLR IHPDGHVDGI REKSDPHIKL QLQAEERGVV


121
SIKGVCANRY LAMKEDGRLL ALKCVTEECF FFERLESNNY NTYRSRKYSN WYVALKRTGQ


181
YKLGSKTGPG QKAILFLPMS AKS


 







Amino acid sequence of Monodelphis domestica (gray short-tailed


opossum) FGF2 (SEQ ID NO: 149) (GenBank accession no. NP_001029148,


which is hereby incorporated by reference in its entirety):








1
MAAGSITTLP ALSGDGGGGG AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGIREKSDPN


61
IKLQLQAEER GVVSIKGVCA NRYLAMKEDG RLLALKYVTE ECFFFERLES NNYNTYRSRK


121
YSNWYVALKR TGQYKLGSKT GPGQKAILFL PMSAKS


 







Amino acid sequence of Oryctolagus cuniculus (rabbit) FGF2 (SEQ ID


NO: 150) (GenBank accession no. XP_002717284, which is hereby


incorporated by reference in its entirety):








1
MAAESITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI


61
KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY


121
SSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS


 







Amino acid sequence of Meleagris gallopavo (turkey) FGF2 (partial


amino acid sequence corresponding to human FGF2 residues 31 to 155)


(SEQ ID NO: 151) (Ensembl accession no. ENSMGAP00000010977, which is


hereby incorporated by reference in its entirety):








1
RLYCKNGGFF LRINPDGRVD GVREKSDPHI KLQLQAEERG VVSIKGVSAN RFLAMKEDGR


61
LLALKCATEE CFFFERLESN NYNTYRSRKY SDWYVALKRT GQYKPGPKTG PGQKAILFLP


121
MSAKS


 







Amino acid sequence of Gallus gallus (chicken) FGF2 (SEQ ID NO: 152)


(GenBank accession no. NP_990764








1
maagaagsit tlpalpddgg ggafppghfk dpkrlyckng gfflrinpdg rvdgvreksd


61
PHIKLQLQAE ERGVVSIKGV SANRFLAMKE DGRLLALKCA TEECFFFERL ESNNYNTYRS


121
RKYSDWYVAL KRTGQYKPGP KTGPGQKAIL FLPMSAKS


 







Amino acid sequence of Taeniopygia guttata (zebra finch) FGF2


(SEQ ID NO: 153) (GenBank accession no. XP_002188397,


which is hereby incorporated by reference in its entirety):








1
MAAAGGIATL PDDGGSGAFP PGHFKDPKRL YCKNGGFFLR INPDGKVDGV REKSDPHIKL


61
QLQAEERGVV SIKGVSANRF LAMKEDGRLL ALKYATEECF FFERLESNNY NTYRSRKYSD


121
WYVALKRTGQ YKPGPKTGPG QKAILFLPMS AKS


 







Amino acid sequence of Cynops pyrrhogaster (Japanese firebelly newt)


FGF2 (SEQ ID NO: 154) (GenBank accession no. BAB63249, which


is hereby incorporated by reference in its entirety):








1
MAAGSITSLP ALPEDGNGGT FTPGGFKEPK RLYCKNGGFF LRINSDGKVD GAREKSDSYI


61
KLQLQAEERG VVSIKGVCAN RYLAMKDDGR LMALKWITDE CFFFERLESN NYNTYRSRKY


121
SDWYVALKRT GQYKNGSKTG AGQKAILFLP MSAKS


 







Amino acid sequence of Xenopus laevis (African clawed frog) FGF2


(SEQ ID NO: 155) (GenBank accession no. NP_001093341,


which is hereby incorporated by reference in its entirety):








1
MAAGSITTLP TESEDGGNTP FSPGSFKDPK RLYCKNGGFF LRINSDGRVD GSRDKSDSHI


61
KLQLQAVERG VVSIKGITAN RYLAMKEDGR LTSLRCITDE CFFFERLEAN NYNTYRSRKY


121
SSWYVALKRT GQYKNGSSTG PGQKAILFLP MSAKS


 







Amino acid sequence of Didelphis albiventris (white-eared opossum)


FGF2 (SEQ ID NO: 156) (GenBank accession no. ABL77404,


which is hereby incorporated by reference in its entirety):








1
MAAGSITTLP ALSGDGGGGG AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGIREKSDPN


61
IKLQLQAEER GVVSIKGVCA NRYLAMKEDG RLLALKYVTE ECFFFERLES NNYNTYRSRK


121
YSNWYVALKR TGQYKLGSKT GPGQKAILFS PCLLRC


 







Amino acid sequence of Myotis lucifugus (microbat) FGF2 (partial


amino acid sequence corresponding to human FGF2 residues 60


to 155) (SEQ ID NO: 157) (Ensembl accession no. ENSMLUP00000017859,


which is hereby incorporated by reference in its entirety):








1
VKLQLQAEER GVVSIKGVCA NRYLAMKEDG RLQASKCVTD ECFFFERLES NNYNTYRSRK


61
YSSWYVALKR NGQYKLGPKT GPGQKAILFL PMSAKS


 







Amino acid sequence of Anolis carolinensis (anole lizard) FGF2


(partial amino acid sequence corresponding to human FGF2 residues 16


to 155) (SEQ ID NO: 158) (Ensembl accession no. ENSACAP00000011657,


which is hereby incorporated by reference in its entirety):








1
AAAASFPPGP FKDPKRLYCK NGGFFLRINP DGGVDGVREK SDPNIKLLLQ AEERGVVSIK


61
GVCANRFLAM NEDGRLLALK YVTDECFFFE RLESNNYNTY RSRKYRDWYI ALKRTGQYKL


121
GPKTGRGQKA ILFLPMSAKS


 







Amino acid sequence of Dasypus novemcinctus (armadillo) FGF2


(partial amino acid sequence corresponding to human FGF2 residues


1 to 94) (SEQ ID NO: 159) (Ensembl accession no. ENSDNOP00000011351,


which is hereby incorporated by reference in its entirety):








124
   MAAGSIT TLPALPEDGG SGAFPPGHFK DPKRLYCKNG GFFLRIHPDG RVDGVREKSD


181
PNIKLQLQAE ERGVVSIKGV CANRYLAMRE DGRLQAS


 







Amino acid sequence of Tupaia belangeri (tree shrew) FGF2(SEQ ID


NO: 160) (Ensembl accession no. ENSTBEP00000000985, which is hereby


incorporated by reference in its entirety):








1
AGVRAEREEA PGSGDSRGTD PAARSLIRRP DAAAREALLG ARSRVQGSST SWPASSRTGI


61
KLPDDSGQGM GGYPLDRPSR STGRGLGGAP DPAVKLQLQA EERGVVSIKG VCANRYLAMK


121
EDGRLLASKC VTDECFFFER LESNNYNTYR SRKYSSWYVA LKRTGQYKLG SKTGPGQKAI


181
LFLPMSAKS


 







Amino acid sequence of Xenopus silurana tropicalis (western clawed


frog) FGF2 (SEQ ID NO: 161) (GenBank accession no. NP_001017333,


which is hereby incorporated by reference in its entirety):








1
MAAGSITTLP TESEDGNTPF PPGNFKDPKR LYCKNGGYFL RINSDGRVDG SRDKSDLHIK


61
LQLQAVERGV VSIKGITANR YLAMKEDGRL TSLKCITDEC FFYERLEANN YNTYRSRKNN


121
SWYVALKRTG QYKNGSTTGP GQKAILFLPM SAKS


 







Amino acid sequence of Latimeria chalumnae (coelacanth) FGF2 (SEQ ID


NO: 162) (Ensembl accession no. ENSLACP00000019200, which is hereby


incorporated by reference in its entirety):








1
MAAGGITTLP AVPEDGGSST FPPGNFKEPK RLYCKNGGYF LRINPDGRVD GTREKNDPYI


61
KLQLQAESIG VVSIKGVCSN RYLAMNEDCR LFGLKYPTDE CFFHERLESN NYNTYRSKKY


121
SDWYVALKRT GQYKPGPKTG LGQKAILFLP MSAKS


 







Amino acid sequence of Tetraodon nigroviridis (spotted green


pufferfish) FGF2 (SEQ ID NO: 163) (GenBank accession no. CAG04681,


which is hereby incorporated by reference in its entirety):








34
                                    MATGGIT TLPSTPEDGG SSGFPPGSFK


61
DPKRLYCKNG GFFLRIKSDG VVDGIREKSD PHIKLQLQAT SVGEVVIKGV CANRYLAMNR


121
DGRLFGTKRA TDECHFLERL ESNNYNTYRS RKYPTMFVGL TRTGQYKSGS KTGPGQKAIL


181
FLPMSAKC


 







Amino acid sequence of Gasterosteus aculeatus (stickleback) FGF2


(SEQ ID NO: 164) (Ensembl accession no. ENSGACP00000022078,


which is hereby incorporated by reference in its entirety):








1
MATAGFATLP STPEDGGSGG FTPGGFKDPK RLYCKNGGFF LRIRSDGGVD GIREKSDAHI


61
KLQIQATSVG EVVIKGVCAN RYLAMNRDGR LFGVRRATDE CYFLERLESN NYNTYRSRKY


121
PGMYVALKRT GQYKSGSKTG PGQKAILFLP MSAKC


 







Amino acid sequence of Takifugu rubripes (fugu rubripes) FGF2


(SEQ ID NO: 165) (GenBank accession no. CAD19830, which is


hereby incorporated by reference in its entirety):








1
MATGGITTLP STPEDGGSGG FPPGSFKDPK RLYCKNGGFF LRIRSDGAVD GTREKTDPHI


61
KLQLQATSVG EVVIKGVCAN RYLAMNRDGR LFGMKRATDE CHFLERLESN NYNTYRSRKY


121
PNMFVGLTRT GNYKSGTKTG PCQKAILFLP MSAKY


 







Amino acid sequence of Oncorhynchus mykiss (rainbow trout) FGF2 (SEQ


ID NO: 166) (GenBank accession no. NP_001118008, which is hereby


incorporated by reference in its entirety):








1
MATGEITTLP ATPEDGGSGG FLPGNFKEPK RLYCKNGGYF LRINSNGSVD GIRDKNDPHN


61
KLQLQATSVG EVVIKGVSAN RYLAMNADGR LFGPRRTTDE CYFMERLESN NYNTYRSRKY


121
PEMYVALKRT GQYKSGSKTG PGQKAILFLP MSARR


 







Amino acid sequence of Salmo salar (salmon) FGF2 (SEQ ID NO: 167)


(GenBank accession no. ACJ02099, which is hereby incorporated by


reference in its entirety):








1
MATGEITTLP ATPEDGGSGG FPPGNFKDPK RLYCKNGGYF LRINSNGSVD GIREKNDPHK


61
QPQFVRAWTL QGVKRSTGML AHVDSNASHN CVKVAGCSLG EFGSMSNRPH NRRPRVATPA


121
QDLHIRLLHL RDRLKPATRT ADKTEEYFCL


 







Amino acid sequence of Danio rerio (zebrafish) FGF2 (SEQ ID NO: 168)


(GenBank accession no. AAP32155, which is hereby incorporated by


reference in its entirety):








1
MATGGITTLP AAPDAENSSF PAGSFRDPKR LYCKNGGFFL RINADGRVDG ARDKSDPHIR


61
LQLQATAVGE VLIKGICTNR FLAMNADGRL FGTKRTTDEC YFLERLESNN YNTYRSRKYP


121
DWYVALKRTG QYKSGSKTSP GQKAILFLPM SAKC


 







Amino acid sequence of Oreochromis niloticus (Nile tilapia) FGF2


(SEQ ID NO: 169) (GenBank accession no. XP_003443412,


which is hereby incorporated by reference in its entirety):








1
MATGGITTLP ATPEDGGSSG FPPGNFKDPK RLYCKNGGFF LRIKSDGGVD GIREKNDPHI


61
KLQLQATSVG EVVIKGICAN RYLAMNRDGR LFGARRATDE CYFLERLESN NYNTYRSRKY


121
PNMYVALKRT GQYKSGSKTG PGQKAILFLP MSAKC


 







Amino acid sequence of Oryzias latipes (medaka) FGF2 (SEQ ID


NO: 170) (Ensembl accession no. ENSORLP00000025834,


which is hereby incorporated by reference in its entirety):








1
MATGEITTLP SPAENSRSDG FPPGNYKDPK RLYCKNGGLF LRIKPDGGVD GIREKKDPHV


61
KLRLQATSAG EVVIKGVCSN RYLAMHGDGR LFGVRQATEE CYFLERLESN NYNTYRSKKY


121
PNMYVALKRT GQYKPGNKTG PGQKAILFLP MSAKY









As noted above, the portion of the paracrine FGF may be modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. In one embodiment, the modification of the paracrine FGF includes one or more substitutions, additions, or deletions.
In one embodiment, the modification is one or more substitutions located at one or more amino acid residues of SEQ ID NO: 121 selected from N36, K128, R129, K134, K138, Q143, K144, C78, C96, and combinations thereof. In one embodiment, the one or more substitutions are selected from N36T, K128D, R129Q, K134V, K138H, Q143M, K144T/L/I, C78S, C96S, and combinations thereof. In one embodiment, the modification is one or more substitutions which are located at one or more amino acid residues corresponding to residues of SEQ ID NO: 121 selected from N36, K128, R129, K134, K138, Q143, K144, C78, C96, and combinations thereof. In one embodiment, the modification is one or more substitutions which are located at one or more amino acid residues corresponding to residues of SEQ ID NO: 121 selected from N36, K128, R129, K134, K138, Q143, K144, C78, C96, and combinations thereof. Amino acid residues corresponding to those of SEQ ID NO: 121 may be determined by, for example, sequence analysis and structural analysis.
It will be understood that the portion of the paracrine FGF according to the present invention may be derived from a nucleotide sequence that encodes a paracrine FGF protein. For example, in one embodiment, nucleotide sequence is the nucleotide sequence that encodes human FGF2 (GenBank Accession No. NM_002006, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 171), as follows:









468
                                                   ATG GCAGCCGGGA



 


481
GCATCACCAC GCTGCCCGCC TTGCCCGAGG ATGGCGGCAG CGGCGCCTTC CCGCCCGGCC


 


541
ACTTCAAGGA CCCCAAGCGG CTGTACTGCA AAAACGGGGG CTTCTTCCTG CGCATCCACC


 


601
CCGACGGCCG AGTTGACGGG GTCCGGGAGA AGAGCGACCC TCACATCAAG CTACAACTTC


 


661
AAGCAGAAGA GAGAGGAGTT GTGTCTATCA AAGGAGTGTG TGCTAACCGT TACCTGGCTA


 


721
TGAAGGAAGA TGGAAGATTA CTGGCTTCTA AATGTGTTAC GGATGAGTGT TTCTTTTTTG


 


781
AACGATTGGA ATCTAATAAC TACAATACTT ACCGGTCAAG GAAATACACC AGTTGGTATG


 


841
TGGCACTGAA ACGAACTGGG CAGTATAAAC TTGGATCCAA AACAGGACCT GGGCAGAAAG


 


901
CTATACTTTT TCTTCCAATG TCTGCTAAGA GCTGA






In another embodiment of the present invention, the portion of the paracrine FGF of the chimeric protein may be derived from a nucleotide sequence that encodes an ortholog of human FGF2. Nucleotide sequences that encode FGF2 orthologs are shown in Table 4.







TABLE 4







Gorilla FGF2 gene coding sequence (amino acids (“aa”) 104-258) (SEQ ID


NO: 172) (Ensembl accession no. ENSGGOT00000004842, which is hereby


incorporated by reference in its entirety):








310
            ATGGCAGCC GGGAGCATCA CCACGCTGCC CGCCTTGCCC GAGGATGGCG


359
GCAGCGGCGC CTTCCCGCCC GGCCACTTCA AGGACCCCAA GCGGCTGTAC TGCAAAAACG


419
GGGGCTTCTT CCTGCGCATC CACCCCGACG GCCGAGTTGA CGGGGTCCGG GAGAAGAGCG


479
ACCCTCACAT CAAGCTACAA CTTCAAGCAG AAGAGAGAGG AGTTGTGTCT ATCAAAGGAG


539
TGTGTGCTAA CCGTTACCTT GCTATGAAGG AAGATGGAAG ATTACTGGCT TCTAAATGTG


599
TTACGGATGA GTGTTTCTTT TTTGAACGAT TGGAATCTAA TAACTACAAT ACTTACCGGT


659
CAAGGAAATA CACCAGTTGG TATGTGGCAC TGAAACGAAC TGGGCAGTAT AAACTTGGAT


719
CCAAAACAGG ACCTGGGCAG AAAGCTATAC TTTTTCTTCC AATGTCTGCT AAGAGCTGA


 







Sumatran orangutan FGF2 gene coding sequence (aa 168-322)


(SEQ ID NO: 173) (GenBank accession no. XM_002815126,


which is hereby incorporated by reference in its entirety):








504
                         ATGGCAG CCGGGAGCAT CACCACGCTG CCCGCCTTGC


541
CCGAGGATGG CGGCAGCGGC GCCTTCCCGC CGGGCCACTT CAAGGACCCC AAGCGGCTGT


601
ACTGCAAAAA CGGGGGCTTC TTCCTGCGCA TCCACCCCGA CGGCCGAGTT GACGGGGTCC


661
GAGAGAAGAG CGACCCTCAC ATCAAACTAC AACTTCAAGC AGAAGAAAGA GGAGTTGTGT


721
CTATCAAAGG AGTGTGTGCT AACCGCTACC TTGCTATGAA GGAAGATGGA AGATTACTGG


781
CTTCTAAATG TGTTACGGAT GAGTGTTTCT TTTTTGAACG ATTGGAATCT AATAACTACA


841
ATACTTACCG GTCAAGGAAA TACACCAGTT GGTATGTGGC ACTGAAACGA ACTGGGCAGT


901
ATAAACTTGG ATCCAAAACA GGACCTGGGC AGAAAGCTAT ACTTTTTCTT CCAATGTCTG


961
CTAAGAGCTG A


 







Rhesus monkey FGF2 gene coding sequence (aa 83-237) (SEQ ID NO: 174)


(GenBank accession no. XM_001099284, which is hereby incorporated by


reference in its entirety):








247
      ATGG CAGCCGGGAG CATCACCACG CTGCCCGCCT TGCCCGAGGA TGGCGGCAGC


301
GGCGCCTTCC CGCCTGGCCA CTTCAAGGAC CCCAAGCGGC TGTACTGCAA AAACGGGGGC


361
TTCTTCCTGC GCATTCACCC CGACGGCCGA GTTGACGGGG TCCGGGAGAA GAGCGACCCT


421
CACATCAAAT TACAACTTCA AGCAGAAGAG AGAGGAGTTG TGTCTATCAA AGGAGTGTGT


481
GCTAACCGTT ACCTTGCTAT GAAGGAAGAT GGAAGATTAC TGGCTTCTAA ATGTGTTACA


541
GATGAGTGTT TCTTTTTTGA ACGATTGGAA TCTAATAACT ACAATACTTA CCGGTCAAGG


601
AAATACACCA GTTGGTATGT GGCACTGAAA CGAACTGGGC AATATAAACT TGGATCCAAA


661
ACAGGACCTG GGCAGAAAGC TATACTTTTT CTTCCAATGT CTGCTAAGAG CTGA


 







Chimpanzee FGF2 gene coding sequence (aa 134-288) (SEQ ID NO: 175)


(GenBank accession no. NM_001110241, which is hereby incorporated by


reference in its entirety):








400
                                          A TGGCAGCCGG GAGCATCACC


421
ACGCTGCCCG CCTTGCCCGA GGATGGCGGC AGCGGCGCCT TCCCGCCCGG CCACTTCAAG


481
GACCCCAAGC GGCTGTACTG CAAAAACGGG GGCTTCTTCC TGCGCATCCA CCCCGACGGC


541
CGAGTTGACG GGGTCCGGGA GAAGAGCGAC CCTCACATCA AGCTACAACT TCAAGCAGAA


601
GAGAGAGGAG TTGTGTCTAT CAAAGGAGTG TGTGCTAACC GTTACCTTGC TATGAAGGAA


661
GATGGAAGAT TACTGGCTTC TAAATGTGTT ACGGATGAGT GTTTCTTTTT TGAACGATTG


721
GAATCTAATA ACTACAATAC TTACCGGTCA AGGAAATACA CCAGTTGGTA TGTGGCACTG


781
AAACGAACTG GGCAGTATAA ACTTGGATCC AAAACAGGAC CTGGGCAGAA AGCTATACTT


841
TTTCTTCCAA TGTCTGCTAA GAGCTGA


 







Pygmy chimpanzee FGF2 gene coding sequence (112-266) (SEQ ID NO: 176)


(GenBank accession no. XM_003816433, which is hereby incorporated by


reference in its entirety):








334
                                    ATGGCAG CCGGGAGCAT CACCACGCTG


361
CCCGCCTTGC CCGAGGATGG CGGCAGCGGC GCCTTCCCGC CCGGCCACTT CAAGGACCCC


421
AAGCGGCTGT ACTGCAAAAA CGGGGGCTTC TTCCTGCGCA TCCACCCCGA CGGCCGAGTT


481
GACGGGGTCC GGGAGAAGAG CGACCCTCAC ATCAAGCTAC AACTTCAAGC AGAAGAGAGA


541
GGAGTTGTGT CTATCAAAGG AGTGTGTGCT AACCGTTACC TTGCTATGAA GGAAGATGGA


601
AGATTACTGG CTTCTAAATG TGTTACGGAT GAGTGTTTCT TTTTTGAACG ATTGGAATCT


661
AATAACTACA ATACTTACCG GTCAAGGAAA TACACCAGTT GGTATGTGGC ACTGAAACGA


721
ACTGGGCAGT ATAAACTTGG ATCCAAAACA GGACCTGGGC AGAAAGCTAT ACTTTTTCTT


781
CCAATGTCTG CTAAGAGCTG A


 







Bolivian squirrel monkey FGF2 gene coding sequence (1-155)


(SEQ ID NO: 177) (GenBank accession no. XM_003936241,


which is hereby incorporated by reference in its entirety):








23
                        ATGGCAGC CGGGAGCATC ACCACGCTGC CCGCCCTGCC


61
CGAAGACGGC GGCAGCGGCG CCTTCCCGCC CGGCCACTTC AAAGACCCCA AGCGGCTGTA


121
CTGCAAAAAC GGGGGCTTCT TCCTGCGAAT CCACCCCGAC GGCCGAGTGG ACGGGGTCCG


181
GGAGAAGAGC GACCCTCACA TCAAACTACA ACTTCAAGCA GAAGAGAGAG GAGTTGTATC


241
TATCAAAGGA GTGTGTGCTA ACCGTTACCT TGCTATGAAG GAAGATGGAA GATTACTGGC


301
TTCTAAATGT GTTACGGACG AGTGTTTCTT TTTTGAACGA TTGGAATCTA ATAACTACAA


361
TACTTACCGA TCAAGGAAAT ACACCAGTTG GTATGTGGCA CTGAAACGAA CTGGGCAGTA


421
TAAACTTGGA TCCAAAACAG GACCTGGGCA GAAAGCTATA CTTTTTCTTC CAATGTCTGC


481
TAAGAGCTGA


 







Northern white-cheeked gibbon FGF2 gene coding sequence (aa 1-155)


(SEQ ID NO: 178) (GenBank accession no. XM_003271356, which is


hereby incorporated by reference in its entirety):








435
                                                   ATG GCAGCCGGGA


481
GCATCACCAC GCTGCCCGCC TTGCCGGAGG ATGGCGGCAG CGGCGCCTTC CCGCCCGGCC


541
ACTTCAAGGA CCCCAAGCGG CTGTACTGCA AAAACGGGGG TTTCTTCCTG CGCATCCACC


601
CCGACGGTCG AGTTGACGGG GTCCGGGAGA AGAGCGACCC TCACATCAAA CTACAACTTC


661
AAGCAGAAGA GAGAGGAGTT GTGTCTATCA AAGGAGTGTG TGCTAACCGT TACCTTGCTA


721
TGAAGGAAGA TGGAAGATTA CTGGCTTCTA AATGTGTTAC GGATGAGTGT TTCTTTTTTG


781
AACGATTGGA ATCTAATAAC TACAATACTT ACCGGTCAAG GAAATACACC AGTTGGTATG


841
TGGCACTGAA ACGAACTGGG CAGTATAAAC TTGGATCCAA AACAGGACCT GGGCAGAAAG


901
CTATACTTTT TCTTCCAATG TCTGCTAAGA GCTGA


 







Horse FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 179) (GenBank


accession no. NM_001195221, which is hereby incorporated by reference in


its entirety):








54
                                                          ATGGCAG


61
CCGGGAGCAT CACCACGCTG CCCGCCCTGC CCGAGGACGG CGGCAGCGGC GCCTTCCCGC


121
CCGGCCACTT CAAGGACCCC AAGCGGCTCT ACTGCAAAAA CGGGGGCTTC TTCCTGCGCA


181
TCCACCCCGA CGGCCGAGTG GACGGGGTCC GGGAGAAGAG CGACCCTCAC ATCAAACTAC


241
AACTTCAAGC AGAAGAGAGA GGGGTTGTGT CTATCAAAGG AGTGTGTGCG AACCGTTATC


301
TTGCTATGAA GGAAGATGGA AGGTTACTGG CTTCTAAATG TGTTACGGAC GAGTGTTTCT


361
TTTTTGAACG ATTGGAATCT AATAACTACA ATACTTACCG GTCAAGGAAA TACTCCAGTT


421
GGTATGTGGC CCTGAAACGA ACGGGGCAGT ATAAACTTGG ACCCAAAACA GGACCTGGAC


481
AGAAAGCTAT ACTTTTTCTT CCAATGTCTG CTAAGAGCTG A


 







Cattle FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 180) (GenBank


accession no. NM_174056, which is hereby incorporated by reference


in its entirety):








104
                                               ATGGCCG CCGGGAGCAT


121
CACCACGCTG CCAGCCCTGC CGGAGGACGG CGGCAGCGGC GCTTTCCCGC CGGGCCACTT


181
CAAGGACCCC AAGCGGCTGT ACTGCAAGAA CGGGGGCTTC TTCCTGCGCA TCCACCCCGA


241
CGGCCGAGTG GACGGGGTCC GCGAGAAGAG CGACCCACAC ATCAAACTAC AACTTCAAGC


301
AGAAGAGAGA GGGGTTGTGT CTATCAAAGG AGTGTGTGCA AACCGTTACC TTGCTATGAA


361
AGAAGATGGA AGATTACTAG CTTCTAAATG TGTTACAGAC GAGTGTTTCT TTTTTGAACG


421
ATTGGAGTCT AATAACTACA ATACTTACCG GTCAAGGAAA TACTCCAGTT GGTATGTGGC


481
ACTGAAACGA ACTGGGCAGT ATAAACTTGG ACCCAAAACA GGACCTGGGC AGAAAGCTAT


541
ACTTTTTCTT CCAATGTCTG CTAAGAGCTG A


 







Olive baboon FGF2 gene coding sequence (1-155) (SEQ ID NO: 181) (GenBank


accession no. XM_003899161, which is hereby incorporated by reference in


its entirety):








467
                                                  ATGG CAGCCGGGAG


481
CATCACCACG CTGCCCGCCT TGCCCGAGGA TGGCGGCAGC GGCGCCTTCC CGCCCGGCCA


541
CTTCAAGGAC CCCAAGCGGC TGTACTGCAA AAACGGGGGC TTCTTCCTGC GCATTCACCC


601
CGACGGCCGA GTTGACGGGG TCCGGGAGAA GAGCGACCCT CACATCAAAT TACAACTTCA


661
AGCAGAAGAG AGAGGAGTTG TGTCTATCAA AGGAGTGTGT GCTAACCGTT ACCTTGCTAT


721
GAAGGAAGAT GGAAGATTAC TGGCTTCTAA ATGTGTTACG GATGAGTGTT TCTTTTTTGA


781
ACGATTGGAA TCTAATAACT ACAATACTTA CCGGTCAAGG AAATACACCA GTTGGTATGT


841
GGCACTGAAA CGAACTGGGC AGTATAAACT TGGATCCAAA ACAGGACCTG GGCAGAAAGC


901
TATACTTTTT CTTCCAATGT CTGCTAAGAG CTGA


 







Alpaca FGF2 gene coding sequence (aa 111-265) (SEQ ID NO: 182) (Ensembl


accession no. ENSVPAT00000010536, which is hereby incorporated by


reference in its entirety):








341
                                 ATGGCAGCTG GGAGCATCAC CACGCTGCCC


361
GCCCTGCCGG AGGACGGCGG CAGCGGCGCC TTCCCGCCCG GCCACTTCAA GGACCCCAAG


421
CGGTTGTACT GCAAAAACGG GGGCTTCTTC CTGCGCATCC ACCCCGACGG CCGAGTGGAC


481
GGGGTCCGGG AGAAGAGCGA CCCTCACATC AAACTACAAC TTCAAGCAGA AGAGAGAGGG


541
GTCGTGTCTA TCAAAGGAGT GTGTGCAAAC CGTTACCTTG CTATGAAGGA AGATGGAAGA


601
TTACTGGCTT CTAAATGTGT CACAGACGAG TGTTTCTTTT TTGAACGATT GGAATCTAAT


661
AACTACAATA CTTACCGGTC AAGGAAATAC TCCAGTTGGT ATGTGGCACT GAAACGAACT


721
GGGCAGTACA AACTTGGACC CAAAACAGGA CCTGGGCAGA AAGCTATACT TTTCCTTCCA


781
ATGTCTGCTA AGAGCTGA


 







Sheep FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 183) (GenBank


accession no. NM_001009769, which is hereby incorporated by reference in


its entirety):








1
ATGGCCGCCG GGAGCATCAC CACGCTGCCA GCCCTGCCGG AGGACGGCGG CAGCAGCGCT


61
TTCCCGCCCG GCCACTTTAA GGACCCCAAG CGGCTGTACT GCAAGAACGG GGGCTTCTTC


121
CTGCGCATCC ACCCCGACGG CCGAGTGGAC GGGGTCCGCG AGAAGAGCGA CCCTCACATC


181
AAACTACAAC TTCAAGCAGA AGAGAGAGGG GTTGTGTCTA TCAAAGGAGT GTGTGCAAAC


241
CGTTACCTTG CTATGAAAGA AGATGGAAGA TTACTAGCTT CTAAATGTGT TACAGACGAG


301
TGTTTCTTTT TTGAACGATT GGAGTCTAAT AACTACAATA CTTACCGGTC AAGGAAATAC


361
TCCAGTTGGT ATGTGGCACT GAAACGAACT GGGCAGTATA AACTTGGACC CAAAACAGGA


421
CCTGGGCAGA AAGCTATACT TTTTCTTCCA ATGTCTGCTA AGAGCTGA


 







Western roe deer FGF2 gene coding sequence (1-108; partial amino acid


sequence corresponding to human FGF2 residues 42 to 149)


(SEQ ID NO: 184) (GenBank accession no. AF152587, which is


hereby incorporated by reference in its entirety):








1
GCGCATCCAC CCCGACGGCC GAGTGGACGG GGTCCGCGAG AAGAGTGACC CTCACATCAA


61
ACTACAACTT CAAGCAGAAG AGAGAGGGGT TGTGTCTATC AAAGGAGTGT GTGCGAACCG


121
TTATCTTGCT ATGAAAGAAG ACGGAAGATT ATTGGCTTCA AAATGTGTTA CAGACGAATG


181
TTTCTTTTTT GAACGATTGG AGTCTAATAA CTACAATACT TACCGGTCAA GGAAATACTC


241
CAGTTGGTAT GTGGCACTGA AACGAACTGG GCAGTATAAA CTTGGACCCA AAACAGGACC


301
TGGGCAGAAA GCTATACTTT TTCTT


 







Elephant FGF2 gene coding sequence (1-96; partial amino acid sequence


corresponding to human FGF2 residues 60 to 155) (SEQ ID NO: 185)


(Ensembl accession no. ENSLAFT00000008249, which is hereby


incorporated by reference in its entirety):








1
GTTAAACTAC AGCTTCAAGC AGAAGAGAGA GGTGTTGTGT CTATCAAAGG AGTGTGTGCC


61
AACCGTTATC TGGCTATGAA GGAAGATGGA AGATTGCTGG CTTCTAGATG TGTGACAGAT


121
GAATGTTTCT TCTTTGAACG ACTGGAATCT AATAACTACA ATACTTACCG GTCAAGGAAA


181
TACACCAGTT GGTATGTGGC ACTGAAACGA ACGGGGCAGT ATAAACTTGG ATCCAAAACA


241
GGACCTGGAC AGAAAGCTAT ACTTTTTCTT CCCATGTCTG CTAAGAGC


 







Pig FGF2 gene coding sequence (1-120; partial amino acid sequence


corresponding to human FGF2 residues 36 to 155) (SEQ ID NO: 186)


(GenBank accession no. AJ577089 and Ensembl accession no.


ENSSSCT00000009952, which is hereby incorporated


by reference in its entirety):








1
GAACGGGGGC TTCTTCCTGC GCATCCACCC CGACGGCCGA GTGGATGGGG TCCGGGAGAA


61
GAGCGACCCT CACATCAAAC TACAACTTCA AGCAGAAGAG AGAGGGGTTG TGTCTATCAA


121
AGGAGTGTGT GCAAACCGTT ATCTTGCTAT GAAGGAAGAT GGAAGATTAC TGGCTTCTAA


181
ATGTGTTACA GACGAGTGTT TCTTTTTTGA ACGACTGGAA TCTAATAACT ACAATACTTA


241
CCGGTCGAGG AAATACTCCA GTTGGTATGT GGCACTGAAA CGAACTGGGC AGTATAAACT


301
TGGACCCAAA ACAGGACCTG GGCAGAAAGC TATACTTTTT CTTCCAATGT CTGCTAAGAG


361
C


 







Panda FGF2 gene coding sequence (1-96; partial amino acid sequence


corresponding to human FGF2 residues 60 to 155) (SEQ ID NO: 187)


(Ensembl accession no. ENSAMET00000019232, which is hereby


incorporated by reference in its entirety):








1
GTCAAACTGC AACTTCAAGC GGAAGAGAGA GGGGTTGTAT CCATCAAAGG AGTATGTGCA


61
AATCGCTATC TTGCCATGAA GGAAGATGGA AGATTACTGG CTTCTAAATG TGTTACCGAT


121
GAGTGTTTCT TTTTTGAGCG ACTGGAATCT AATAACTACA ATACTTACCG GTCAAGGAAA


181
TACTCCAGTT GGTATGTGGC ACTGAAACGA ACTGGGCAGT ATAAACTTGG ACCCAAAACA


241
GGACCTGGGC AGAAAGCTAT ACTTTTTCTT CCAATGTCTG CTAAGAGC


 







Sloth FGF2 gene coding sequence (aa 14-168) (SEQ ID NO: 188) (Ensembl


accession no. ENSCHOT00000011394, which is hereby incorporated by


reference in its entirety):








40
                                          A TGGCAGCCGG GAGCATCACC


61
ACGCTGCCCG CCCTGCCCGA GGACGGAGGC AGCGGCGCCT TACCGCCCGG CCACTTCAAA


121
GATCCCAAGC GGCTCTACTG CAAAAACGGG GGCTTCTTCC TGCGTATCCA TCCCGACGGC


181
AGAGTGGACG GGGTCCGGGA GAAGAGCGAC CCCCACATCA AACTACAACT TCAAGCAGAA


241
GAGAGAGGGG TTGTGTCTAT CAAAGGTGTG TGTGCAAACC GATATCTTGC TATGAAGGAA


301
GATGGAAGAT TACAGGCTTC TAAATGTGTA ACGGACGAGT GTTTCTTTTT TGAACGATTG


361
GAATCTAATA ACTACAATAC GTACCGATCA AGGAAATACT CCAGTTGGTA TGTGGCACTG


421
AAACGAACTG GGCAATATAA ACTTGGACCC AAAACAGGAC CTGGGCAGAA AGCCATACTT


481
TTTCTTCCAA TGTCTGCTAA GAGCTGA


 







Water buffalo FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 189)


(GenBank accession no. JQ326277, which is hereby incorporated by


reference in its entirety):








1
ATGGCCGCCG GGAGCATCAC CACGCTGCCA CCCCTGCCGG AGGACGGCGG CAGCGGCGCT


61
TTCCCGCCCG GCCACTTCAA GGACCCCAAG CGGCTGTACT GCAAGAACGG GGGCTTCTTC


121
CTGCGCATCC ACCCCGACGG CCGAGTGGAC GGGGTCCGCG AGAAGAGCGA CCCACACATC


181
AAACTACAAC TTCAAGCAGA AGAGAGAGGG GTTGTGTCTA TCAAAGGAGT GTGTGCAAAC


241
CGTTACCTTG CTATGAAAGA AGATGGAAGA TTACTAGCTT CCAAATGTGT TACAGACGAG


301
TGTTTCTTTT TTGAACGATT GGAGTCTAGT AACTACAATA CTTACCGGTC AAGGAAATAC


361
TCCAGTTGGT ATGTGGCACT GAAACGAACT GGGCAGTATA AACTTGGACC CAAAACAGGA


421
CCTGGGCAGA AAGCTATACT TTTTCTTCCA ATGTCTGCTA AGAGCTGA


 







Dog FGF2 gene coding sequence (aa 40-194) (SEQ ID NO: 190) (GenBank


accession no. XM_003432481, which is hereby incorporated by reference in


its entirety):








118
                                                              ATG


121
GCAGCCGGGA GCATCACCAC GCTGCCCGCC CTGCCGGAGG ACGGCGGCAG CGGCGCCTTC


181
CCGCCCGGCC ACTTCAAGGA CCCCAAGAGG CTGTACTGCA AAAAAGGGGG CTTCTTCCTG


241
CGGATCCACC CCGACGGCCG GGTGGACGGG GTCCGGGAGA AGAGCGATCC CCACGTCAAA


301
TTGCAACTTC AAGCAGAAGA GAGAGGCGTT GTGTCCATCA AAGGAGTATG TGCAAATCGC


361
TATCTTGCTA TGAAGGAAGA TGGAAGATTA CTGGCTTCTA AATGTGTTAC TGACGAGTGC


421
TTCTTTTTTG AACGATTGGA ATCTAATAAC TACAATACTT ACCGGTCAAG GAAATACTCC


481
AGTTGGTATG TGGCACTGAA ACGAACTGGG CAGTATAAAC TTGGACCAAA AACAGGACCT


541
GGGCAGAAAG CTATACTTTT TCTTCCAATG TCTGCTAAGA GCTGA


 







Norway rat FGF2 gene coding sequence (aa 1-154) (SEQ ID NO: 191)


(GenBank accession no. NM_019305, which is hereby


incorporated by reference in its entirety):








533
                                                         ATGGCTGC


541
CGGCAGCATC ACTTCGCTTC CCGCACTGCC GGAGGACGGC GGCGGCGCCT TCCCACCCGG


601
CCACTTCAAG GATCCCAAGC GGCTCTACTG CAAGAACGGC GGCTTCTTCC TGCGCATCCA


661
TCCAGACGGC CGCGTGGACG GCGTCCGGGA GAAGAGCGAC CCACACGTCA AACTACAGCT


721
CCAAGCAGAA GAGAGAGGAG TTGTGTCCAT CAAGGGAGTG TGTGCGAACC GGTACCTGGC


781
TATGAAGGAA GATGGACGGC TGCTGGCTTC TAAGTGTGTT ACAGAAGAGT GTTTCTTCTT


841
TGAACGCCTG GAGTCCAATA ACTACAACAC TTACCGGTCA CGGAAATACT CCAGTTGGTA


901
TGTGGCACTG AAACGAACTG GGCAGTATAA ACTCGGATCC AAAACGGGGC CTGGACAGAA


961
GGCCATACTG TTTCTTCCAA TGTCTGCTAA GAGCTGA


 







Naked mole-rat FGF2 gene coding sequence (1-134; partial amino acid


sequence corresponding to human FGF2 residues 22 to 155)


(SEQ ID NO: 192) (GenBank accession no. JH173674, which


is hereby incorporated by reference in its entirety):








378500
                    C CACCCGGCCA CTTCAAGGAC CCAAAGCGGC


378531
TGTACTGCAA AAACGGGGGC TTCTTCCTGC GCATCCACCC CGACGGCCGC


378581
GTGGACGGGG TCCGGGAGAA GAGCGACCCT CACG


418784
   TCAAACT ACAACTTCAA GCAGAAGAGA GAGGAGTTGT GTCTATTAAG


418831
GGAGTGTGTG CGAACCGTTA CCTTGCTATG AAGGAAGATG GAAGATTACT


418881
GGCTTCT


433983
  AAATGTGT TACAGATGAG TGTTTCTTTT TTGAACGATT GGAATCTAAT


434031
AACTACAATA CTTATCGGTC AAGGAAATAC TCCAGTTGGT ATGTGGCACT


434081
GAAACGAACT GGACAATATA AACTTGGATC CAAAACAGGA CCGGGGCAGA


434131
AAGCTATACT TTTTCTTCCA ATGTCTGCTA AGAGCTGA


 







Bushbaby FGF2 gene coding sequence (aa 52-206) (SEQ ID NO: 193) (Ensembl


accession no. ENSOGAT00000025228, which is hereby incorporated by


reference in its entirety):








154
                                    ATGGCAG CCGGGAGCAT CACCACGCTG


181
CCCTCCCTGC CCGAGGACGG CGGCAGCGAC GCCTTTCCGC CCGGCCACTT CAAGGACCCC


241
AAGCGACTGT ACTGCAAAAA CGGGGGCTTC TTCCTGCGCA TCCACCCCGA CGGCCGAGTG


301
GACGGGGTCC GGGAGAAGAG CGACCCTTAC ATCAAACTAC AACTTCAAGC AGAAGAGAGA


361
GGAGTTGTGT CTATCAAAGG AGTGTGTGCG AACCGTTACC TTGCTATGAA GGAAGACGGA


421
AGATTGCTGG CTTCTAAATT GATTACAGAC GAGTGCTTCT TTTTTGAACG ACTGGAATCT


481
AATAACTACA ATACTTACCG GTCAAGAAAA TACTCCAGTT GGTATGTGGC ACTGAAACGA


541
ACTGGACAGT ATAAACTTGG ATCCAAAACA GGACCTGGGC AGAAAGCTAT ACTTTTTCTT


601
CCAATGTCTG CTAAGAGCTG A


 







House mouse FGF2 gene coding sequence (aa 1-154) (SEQ ID NO: 194)


(GenBank accession no. NM_008006, which is hereby


incorporated by reference in its entirety):








198
                  ATG GCTGCCAGCG GCATCACCTC GCTTCCCGCA CTGCCGGAGG


241
ACGGCGGCGC CGCCTTCCCA CCAGGCCACT TCAAGGACCC CAAGCGGCTC TACTGCAAGA


301
ACGGCGGCTT CTTCCTGCGC ATCCATCCCG ACGGCCGCGT GGATGGCGTC CGCGAGAAGA


361
GCGACCCACA CGTCAAACTA CAACTCCAAG CAGAAGAGAG AGGAGTTGTG TCTATCAAGG


421
GAGTGTGTGC CAACCGGTAC CTTGCTATGA AGGAAGATGG ACGGCTGCTG GCTTCTAAGT


481
GTGTTACAGA AGAGTGTTTC TTCTTTGAAC GACTGGAATC TAATAACTAC AATACTTACC


541
GGTCACGGAA ATACTCCAGT TGGTATGTGG CACTGAAACG AACTGGGCAG TATAAACTCG


601
GATCCAAAAC GGGACCTGGA CAGAAGGCCA TACTGTTTCT TCCAATGTCT GCTAAGAGCT


661
GA


 







Squirrel FGF2 gene coding sequence (1-144; partial amino acid sequence


corresponding to human FGF2 residues 12 to 155) (SEQ ID NO: 195)


(Ensembl accession no. ENSSTOT00000022105, which is


hereby incorporated by reference in its entirety):








1
CTGCCCGAGG ACGGCGGCGG CGGCGCCTTC CCGCCCGGCC ACTTTAAGGA CCCCAAGCGG


61
CTCTACTGCA AAAACGGAGG CTTCTTCCTG CGCATCCACC CCGACGGCCG AGTGGACGGG


121
GTCCGGGAGA AGAGCGACCC CCACATCAAG CTCCAGCTTC AAGCCGAAGA CCGAGGGGTT


181
GTGTCCATCA AGGGAGTGTG TGCAAACCGA TACCTGGCCA TGAAGGAGGA CGGGAGGCTC


241
CTGGCTTCTA AATGTGTTAC GGACGAGTGT TTCTTTTTTG AACGACTGGA ATCAAATAAC


301
TACAATACTT ACCGGTCAAG GAAATACTCC AGTTGGTATG TGGCCCTGAA ACGAACAGGG


361
CAGTATAAAC TTGGATCCAA AACAGGACCT GGGCAGAAAG CTATACTTTT TCTTCCAATG


421
TCTGCTAAGA GC


 







Domestic cat FGF2 gene coding sequence (1-106; partial amino acid


sequence corresponding to human FGF2 residues 25 to 130) (SEQ ID


NO: 196) (GenBank accession no. EU314952, which is hereby


incorporated by reference in its entirety):








1
CCACTTCAAG GACCCCAAGC GTCTGTACTG CAAAAACGGG GGCTTCTTCC TGCGCATCCA


61
CCCCGACGGC CGAGTGGATG GGGTCCGGGA GAAGAGCGAC CCTCACATCA AACTGCAACT


121
TCAGGCAGAA GAGAGAGGGG TTGTGTCCAT CAAAGGAGTC TGTGCAAACC GCTATCTTGC


181
CATGAAGGAA GATGGAAGAT TACTGGCTTC TAAATGTGTT ACGGACGAGT GTTTCTTTTT


241
TGAACGATTG GAATCTAATA ACTACAATAC TTATCGGTCA AGGAAATACT CCAGCTGGTA


301
TGTGGCACTG AAACGAAC


 







Guinea pig FGF2 gene coding sequence (1-96; partial amino acid sequence


corresponding to human FGF2 residues 60 to 155) (SEQ ID NO: 197)


(Ensembl accession no. ENSCPOT00000005443, which is hereby


incorporated by reference in its entirety):








1
GTTAAACTAC AACTTCAAGC CGAAGACAGA GGAGTTGTGT CTATCAAGGG AGTCTGTGCG


61
AACCGTTACC TTGCTATGAA GGAAGACGGA AGATTATTGG CTTCCAAATG TGTTACAGAT


121
GAATGTTTCT TTTTTGAACG ACTGGAATCT AATAACTACA ACACTTACCG GTCAAGGAAA


181
TACTCCAGTT GGTATGTGGC ACTGAAACGA ACTGGACAAT ATAAACTTGG GTCCAAAACA


241
GGACCAGGGC AGAAAGCCAT ACTTTTTCTT CCAATGTCTG CGAAGAGC


 







Tasmanian devil FGF2 gene coding sequence (aa 48-203) (SEQ ID NO: 198)


(Ensembl accession no. ENSSHAP00000012215, which is hereby incorporated


by reference in its entirety):








142
                       ATGGCCGCG GGCAGCATCA CCACGTTGCC GGCCCTGGCC


181
GGGGATGGAG CCAGCGGGGG CGCCTTTCCC CCGGGCCACT TCCAGGACCC CAAGCGGCTG


241
TACTGCAAGA ACGGAGGCTT CTTCTTGCGC ATCCATCCCG ACGGTCACGT GGACGGCATC


301
CGCGAGAAGA GCGATCCGCA CATTAAACTT CAGCTTCAGG CAGAAGAGAG AGGAGTAGTG


361
TCTATTAAAG GAGTTTGTGC CAACCGCTAT CTTGCCATGA AAGAGGATGG CAGATTACTG


421
GCTCTGAAAT GTGTGACTGA AGAGTGTTTC TTCTTTGAAC GTCTAGAGTC CAACAATTAC


481
AACACTTATC GCTCAAGGAA ATACTCCAAT TGGTATGTGG CATTGAAACG CACAGGCCAG


541
TATAAGCTTG GATCCAAGAC TGGACCAGGG CAGAAAGCCA TCCTTTTCCT TCCCATGTCT


601
GCTAAGAGCT GA


 







Gray short-tailed opossum FGF2 gene coding sequence (aa 1-155) (SEQ ID


NO: 199) (GenBank accession no. NM_001033976, which is


hereby incorporated by reference in its entirety):








29
                              AT GGCCGCAGGC AGCATCACCA CGCTGCCAGC


61
CCTGTCCGGG GACGGAGGCG GCGGGGGCGC CTTTCCCCCG GGCCACTTCA AGGACCCCAA


121
GCGGCTGTAC TGCAAGAACG GAGGCTTCTT CCTGCGCATC CACCCCGACG GCCGTGTGGA


181
CGGCATCCGC GAGAAGAGCG ACCCGAACAT TAAACTACAA CTTCAGGCAG AAGAGAGAGG


241
AGTGGTGTCT ATTAAAGGAG TATGTGCCAA TCGCTATCTT GCCATGAAGG AAGATGGAAG


301
ATTATTGGCT TTGAAATATG TGACCGAAGA GTGTTTCTTT TTCGAACGCT TGGAGTCCAA


361
CAACTACAAC ACTTATCGCT CGAGGAAATA TTCCAATTGG TACGTGGCAC TGAAACGAAC


421
GGGGCAGTAC AAGCTTGGAT CCAAGACTGG CCCGGGGCAG AAAGCCATCC TTTTCCTCCC


481
CATGTCTGCT AAGAGCTGA


 







Rabbit FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 200) (GenBank


accession no. XM_002717238, which is hereby incorporated by reference in


its entirety):








1
ATGGCAGCCG AGAGCATCAC CACGCTGCCC GCCCTGCCGG AGGATGGAGG CAGCGGCGCC


61
TTCCCGCCCG GCCACTTCAA GGACCCCAAG CGGCTGTACT GCAAAAACGG GGGTTTCTTC


121
CTGCGTATCC ACCCCGACGG CCGCGTGGAC GGGGTCCGGG AGAAGAGCGA CCCACACATC


181
AAATTACAAC TTCAAGCAGA AGAGAGAGGA GTTGTATCCA TCAAAGGTGT GTGTGCAAAC


241
CGTTACCTTG CTATGAAGGA AGATGGAAGA CTGCTGGCTT CTAAATGTGT TACAGACGAG


301
TGCTTCTTTT TTGAACGACT GGAGTCTAAT AACTACAATA CTTACCGGTC AAGGAAATAT


361
TCCAGCTGGT ATGTGGCACT GAAACGAACT GGGCAGTATA AACTTGGATC CAAAACAGGA


421
CCTGGGCAGA AGGCTATACT TTTTCTTCCA ATGTCTGCTA AGAGCTGA


 







Turkey FGF2 gene coding sequence (1-125; partial amino acid sequence


corresponding to human FGF2 residues 31 to 155) (SEQ ID NO: 201)


(Ensembl accession no. ENSMGAT00000011845, which is hereby


incorporated by reference in its entirety):








1
CGGCTCTACT GTAAGAACGG CGGCTTCTTC CTGCGCATCA ATCCCGACGG CAGAGTGGAC


61
GGCGTCCGCG AGAAGAGCGA TCCGCACATC AAACTGCAGC TTCAGGCAGA AGAAAGAGGA


121
GTGGTATCAA TCAAAGGTGT AAGTGCAAAC CGCTTTCTGG CTATGAAGGA GGATGGCAGA


181
TTGCTGGCAC TGAAATGTGC AACAGAAGAA TGTTTCTTTT TTGAGCGTTT GGAATCTAAT


241
AATTATAACA CTTACCGGTC ACGGAAGTAC TCTGATTGGT ATGTGGCACT GAAAAGAACT


301
GGACAGTACA AGCCCGGACC AAAAACTGGA CCTGGACAGA AAGCTATCCT TTTTCTTCCA


361
ATGTCTGCTA AAAGC


 







Gallus gallus FGF2 gene coding sequence (aa 1-158) (SEQ ID NO: 202)


(GenBank accession no. NM_205433, which is hereby incorporated by


reference in its entirety):








98
                                        ATG GCGGCGGGGG CGGCGGGGAG


121
CATCACCACG CTGCCGGCGC TGCCCGACGA CGGGGGCGGC GGCGCTTTTC CCCCCGGGCA


181
CTTCAAGGAC CCCAAGCGGC TCTACTGCAA GAACGGCGGC TTCTTCCTGC GCATCAACCC


241
CGACGGCAGG GTGGACGGCG TCCGCGAGAA GAGCGATCCG CACATCAAAC TGCAGCTTCA


301
AGCAGAAGAA AGAGGAGTAG TATCAATCAA AGGCGTAAGT GCAAACCGCT TTCTGGCTAT


361
GAAGGAGGAT GGCAGATTGC TGGCACTGAA ATGTGCAACA GAGGAATGTT TCTTTTTCGA


421
GCGCTTGGAA TCTAATAACT ATAACACTTA CCGGTCACGG AAGTACTCTG ATTGGTATGT


481
GGCACTGAAA AGGACTGGAC AGTACAAGCC CGGACCAAAA ACTGGACCTG GACAGAAAGC


541
TATCCTTTTT CTTCCAATGT CTGCTAAAAG CTGA


 







Zebra finch FGF2 gene coding sequence (aa 1-153) (SEQ ID NO: 203)


(GenBank accession no. XM_002188361, which is hereby


incorporated by reference in its entirety):








1
ATGGCGGCGG CGGGGGGCAT CGCTACGCTG CCCGACGACG GCGGCAGCGG CGCCTTTCCC


61
CCGGGGCACT TCAAGGACCC CAAGCGCCTG TACTGCAAGA ACGGCGGCTT CTTCCTGCGC


121
ATCAACCCCG ACGGGAAGGT GGACGGCGTC CGCGAGAAGA GCGACCCGCA CATCAAGCTG


181
CAGCTTCAGG CGGAGGAACG AGGAGTGGTG TCCATCAAAG GTGTCAGTGC CAATCGCTTC


241
CTGGCCATGA AAGAGGATGG CAGATTGCTG GCCTTGAAAT ATGCAACAGA AGAATGTTTC


301
TTTTTTGAAC GTTTGGAATC CAATAACTAT AACACTTACC GGTCACGGAA ATACTCGGAT


361
TGGTATGTGG CACTGAAAAG AACTGGACAG TACAAACCTG GACCAAAAAC TGGACCTGGA


421
CAGAAAGCTA TCCTTTTCCT TCCTATGTCT GCTAAAAGCT GA


 







Japanese firebelly newt FGF2 gene coding sequence (aa 1-155) (SEQ ID


NO: 204) (GenBank accession no. AB064664, which is hereby


incorporated by reference in its entirety):








384
                         ATGGCTG CTGGGAGCAT CACCAGTCTC CCTGCCCTAC


421
CCGAGGACGG GAATGGCGGC ACCTTCACAC CCGGCGGATT CAAAGAGCCG AAGAGGCTGT


481
ACTGCAAGAA CGGGGGCTTC TTTCTCCGGA TCAACTCCGA CGGCAAGGTG GACGGAGCCC


541
GGGAGAAGAG CGACTCCTAC ATTAAACTGC AGCTTCAAGC AGAAGAGCGC GGTGTGGTGT


601
CCATCAAGGG AGTATGTGCA AACCGCTATC TCGCTATGAA GGATGATGGC AGGCTGATGG


661
CGCTGAAATG GATAACCGAT GAATGCTTCT TTTTCGAGCG ACTGGAGTCC AACAACTATA


721
ACACGTATCG ATCACGGAAA TATTCCGATT GGTATGTGGC GCTGAAAAGA ACTGGGCAAT


781
ACAAAAATGG ATCAAAAACC GGAGCAGGAC AGAAAGCAAT CCTTTTTCTA CCCATGTCGG


841
CCAAGAGTTG A


 







African clawed frog FGF2 gene coding sequence (aa 1-155)


(SEQ ID NO: 205) (GenBank accession no. NM_001099871,


which is hereby incorporated by reference in its entirety):








335
                                     ATGGCG GCAGGGAGCA TCACAACTCT


361
GCCAACTGAA TCCGAGGATG GGGGAAACAC TCCTTTTTCA CCAGGGAGTT TTAAAGACCC


421
CAAGAGGCTC TACTGCAAGA ACGGGGGCTT CTTCCTCAGG ATAAACTCAG ACGGGAGAGT


481
GGACGGGTCA AGGGACAAAA GTGACTCGCA CATAAAATTA CAGCTACAAG CTGTAGAGCG


541
GGGAGTGGTA TCAATAAAGG GAATCACTGC AAATCGCTAC CTTGCCATGA AGGAAGATGG


601
GAGATTAACA TCGCTGAGGT GTATAACAGA TGAATGCTTC TTTTTTGAAC GACTGGAAGC


661
TAATAACTAC AACACTTACC GGTCTCGGAA ATACAGCAGC TGGTATGTGG CACTAAAGCG


721
AACCGGGCAG TACAAAAATG GATCGAGCAC TGGACCGGGA CAAAAAGCTA TTTTATTTCT


781
CCCAATGTCC GCAAAGAGCT GA


 







White-eared opossum FGF2 gene coding sequence (aa 1-156) (SEQ ID


NO: 206) (GenBank accession no. EF057322, which is hereby


incorporated by reference in its entirety):








1
ATGGCAGCAG GCAGCATCAC CACATTGCCG GCCCTGTCCG GGGACGGAGG CGGCGGGGGA


61
GCCTTTCCTC CAGGCCACTT CAAGGACCCC AAGCGGCTGT ACTGCAAGAA CGGAGGCTTC


121
TTCCTGCGCA TCCACCCCGA CGGCCGCGTG GACGGCATCC GCGAGAAGAG CGACCCGAAC


181
ATTAAACTAC AACTTCAGGC AGAAGAGAGA GGAGTAGTGT CTATTAAAGG AGTATGTGCC


241
AACCGATATC TTGCCATGAA GGAGGATGGC AGATTATTGG CTTTGAAATA TGTGACCGAA


301
GAGTGTTTCT TTTTTGAACG TTTGGAGTCC AACAACTACA ACACTTATCG CTCAAGAAAA


361
TATTCCAATT GGTATGTGGC ACTGAAACGA ACGGGGCAGT ATAAGCTTGG ATCCAAGACT


421
GGCCCGGGGC AGAAAGCCAT CCTTTTCTCC CCATGTCTGC TAAGATGCTG A


 







Microbat FGF2 gene coding sequence (1-96; partial amino acid sequence


corresponding to human FGF2 residues 60 to 155) (SEQ ID NO: 207)


(Ensembl accession no. ENSMLUT00000027717, which is hereby


incorporated by reference in its entirety):








1
GTCAAACTCC AACTTCAAGC AGAAGAGAGA GGGGTCGTGT CTATCAAAGG AGTGTGTGCC


61
AACCGCTATC TCGCTATGAA GGAGGACGGC CGGTTACAGG CTTCTAAATG TGTTACGGAT


121
GAGTGTTTCT TTTTTGAACG GTTGGAATCC AATAACTACA ACACTTACCG GTCAAGAAAG


181
TACTCCAGTT GGTATGTGGC ATTGAAGCGG AATGGGCAGT ATAAACTTGG ACCCAAAACA


241
GGACCTGGCC AGAAAGCCAT ACTTTTTCTT CCCATGTCTG CTAAGAGC


 







Anole lizard FGF2 gene coding sequence (1-140; partial amino acid


sequence corresponding to human FGF2 residues 16 to 155) (SEQ ID


NO: 208) (Ensembl accession no. ENSACAT00000011897, which is


hereby incorporated by reference in its entirety):








1
GCGGCGGCGG CCTCTTTCCC CCCGGGCCCC TTCAAGGACC CCAAGCGCCT CTACTGCAAG


61
AACGGGGGCT TCTTCCTGCG GATCAACCCC GACGGCGGCG TGGACGGCGT CCGAGAGAAG


121
AGCGACCCCA ACATCAAATT GCTGCTCCAG GCAGAGGAGA GAGGTGTAGT GTCCATCAAA


181
GGTGTATGCG CAAACCGTTT CCTGGCTATG AATGAAGACG GTCGATTGTT AGCACTGAAA


241
TACGTAACAG ATGAATGCTT CTTTTTTGAA CGCTTGGAAT CTAATAATTA CAATACTTAT


301
CGGTCTCGTA AATACCGTGA TTGGTACATT GCACTGAAAC GAACTGGTCA GTACAAACTT


361
GGACCAAAAA CTGGACGAGG CCAGAAAGCT ATCCTTTTCC TTCCAATGTC TGCCAAAAGT


 







Armadillo FGF2 gene coding sequence (124-217; partial amino acid


sequence corresponding to human FGF2 residues 1 to 94) (SEQ ID


NO: 209) (Ensembl accession no. ENSDNOT00000014647,


which is hereby incorporated by reference in its entirety):








361
         A TGGCAGCCGG GAGCATCACC ACGCTGCCCG CTCTGCCCGA GGACGGCGGC


421
AGCGGCGCCT TCCCGCCGGG CCACTTCAAG GACCCCAAGC GGCTGTACTG CAAAAACGGG


481
GGCTTCTTCC TGCGCATCCA TCCCGACGGC CGAGTGGACG GGGTCCGGGA GAAGAGCGAC


541
CCTAACATCA AACTACAACT TCAAGCAGAA GAGAGAGGGG TCGTGTCTAT CAAAGGCGTG


601
TGTGCGAACC GTTACCTTGC TATGCGGGAA GACGGAAGAC TCCAGGCGTC T


 







Tree shrew FGF2 gene coding sequence (1-189) (SEQ ID NO: 210) (Ensembl


accession no. ENSTBET00000001143, which is hereby incorporated by


reference in its entirety):








1
GCGGGGGTTA GAGCTGAGAG GGAGGAGGCA CCGGGGAGCG GTGACAGCCG GGGGACCGAT


61
CCCGCCGCGC GTTCGCTCAT CAGGAGGCCG GATGCTGCAG CGCGAGAGGC GCTTCTTGGA


121
GCCAGGAGCC GGGTTCAGGG CAGCTCCACC TCCTGGCCAG CCTCGTCACG AACCGGGATC


181
AAGTTGCCGG ACGACTCAGG TCAAGGAATG GGCGGCTATC CTCTGGACCG CCCGAGCCGG


241
AGCACAGGGC GAGGGCTGGG CGGTGCCCCG GACCCTGCCG TAAAACTACA GCTTCAAGCG


301
GAAGAGAGAG GGGTCGTGTC TATCAAAGGA GTGTGTGCAA ACCGTTACCT GGCCATGAAG


361
GAGGATGGGC GACTGCTGGC TTCTAAATGT GTTACAGATG AGTGTTTCTT TTTTGAACGA


421
CTGGAATCTA ATAACTACAA TACTTACCGG TCCCGAAAGT ACTCCAGCTG GTATGTGGCA


481
CTGAAACGAA CTGGGCAGTA TAAACTTGGA TCCAAAACAG GACCTGGGCA GAAAGCTATA


541
CTTTTTCTTC CAATGTCTGC TAAAAGC


 







Western clawed frog FGF2 gene coding sequence (aa 1-154) (SEQ ID NO:


211) (GenBank accession no. NM_001017333, which is


hereby incorporated by reference in its entirety):








171
                                                       ATGGCAGCAG


181
GAAGCATCAC AACCCTACCA ACCGAATCTG AGGATGGAAA CACTCCTTTC CCACCGGGGA


241
ACTTTAAGGA CCCCAAGAGG CTCTACTGCA AGAATGGGGG CTACTTCCTC AGGATTAACT


301
CAGACGGGAG AGTGGACGGA TCAAGGGATA AAAGTGACTT ACACATAAAA TTACAGCTAC


361
AAGCAGTAGA GCGGGGAGTG GTATCAATAA AGGGAATCAC TGCAAATCGC TACCTTGCCA


421
TGAAGGAAGA TGGGAGATTA ACATCGCTGA AGTGTATAAC AGATGAATGC TTCTTTTATG


481
AACGATTGGA AGCTAATAAC TACAACACTT ACCGGTCTCG GAAAAACAAC AGCTGGTATG


541
TGGCACTAAA GCGAACTGGG CAGTATAAAA ATGGATCGAC CACTGGACCA GGACAAAAAG


601
CTATTTTGTT TCTCCCAATG TCAGCAAAAA GCTGA


 







Coelacanth FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 212)


(Ensembl accession no. ENSLACT00000019333, which is hereby


incorporated by reference in its entirety):








1
                      ATGGCTGCGG GAGGAATCAC TACCCTGCCG GCGGTACCTG


41
AGGATGGAGG CAGCAGCACC TTCCCTCCAG GAAACTTCAA GGAGCCCAAG AGACTTTACT


101
GTAAGAATGG AGGCTATTTC TTAAGGATAA ACCCCGATGG AAGAGTGGAT GGAACAAGGG


161
AGAAAAATGA TCCTTATATA AAATTACAAC TGCAAGCTGA ATCTATAGGA GTGGTGTCGA


221
TAAAGGGAGT TTGTTCAAAC CGTTACCTAG CGATGAATGA AGACTGTAGA CTTTTTGGAT


281
TGAAATATCC AACGGATGAA TGTTTCTTCC ATGAGAGGCT GGAGTCCAAC AACTACAATA


341
CTTATCGTTC AAAGAAGTAT TCGGATTGGT ATGTGGCGCT GAAACGGACT GGTCAGTACA


401
AACCTGGGCC AAAAACTGGA CTGGGACAAA AAGCAATCCT TTTCCTTCCG ATGTCTGCCA


461
AGAGTTGA


 







Spotted green pufferfish FGF2 gene coding sequence (aa 34-188) (SEQ ID


NO: 213) (Ensembl accession no. ENSTNIT00000016254, which is hereby


incorporated by reference in its entirety):








1
ATGGCCACGG GAGGGATCAC GACGCTTCCA TCCACACCTG AAGACGGCGG CAGCAGCGGC


61
TTTCCTCCCG GCAGCTTCAA GGATCCCAAA AGGCTCTACT GTAAAAACGG AGGTTTCTTC


121
CTGAGGATCA AGTCCGACGG GGTCGTGGAC GGAATCCGGG AGAAGAGTGA CCCCCACATA


181
AAGCTTCAGC TCCAGGCGAC CTCTGTGGGG GAGGTGGTCA TCAAGGGGGT GTGCGCTAAC


241
CGCTATCTGG CCATGAACAG AGATGGACGG CTGTTCGGAA CGAAACGAGC CACGGACGAA


301
TGCCATTTCT TAGAGCGGCT TGAGAGCAAC AACTACAACA CTTACCGCTC CAGGAAGTAC


361
CCAACCATGT TTGTGGGACT GACGCGGACG GGCCAGTACA AGTCTGGGAG CAAAACTGGA


421
CCGGGCCAAA AGGCCATCCT TTTTCTTCCG ATGTCCGCCA AATGCTAA


 







Stickleback FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 214)


(Ensembl accession no. ENSGACT00000022120, which is hereby


incorporated by reference in its entirety):








1
                   AT GGCCACGGCA GGCTTCGCGA CGCTTCCCTC CACGCCCGAA


43
GACGGCGGCA GCGGCGGCTT CACCCCCGGG GGATTCAAGG ATCCCAAGAG GCTGTACTGC


103
AAAAACGGGG GCTTCTTCTT GAGGATCAGG TCCGACGGAG GTGTAGATGG AATCAGGGAG


163
AAGAGCGACG CCCACATAAA GCTCCAAATC CAGGCGACGT CGGTGGGGGA GGTGGTCATC


223
AAAGGAGTCT GTGCCAACCG CTATCTGGCC ATGAACAGAG ACGGCCGGCT GTTCGGAGTG


283
AGACGGGCGA CGGACGAATG CTACTTCCTG GAGCGGCTGG AGAGTAACAA CTACAACACC


343
TACCGCTCCA GGAAGTACCC CGGCATGTAC GTGGCTCTGA AGCGGACCGG CCAGTACAAG


403
TCCGGGAGCA AAACCGGACC CGGTCAAAAG GCCATTCTGT TCCTCCCCAT GTCGGCTAAG


463
TGCTAA


 







Fugu rubripes FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 215)


(Ensembl accession no. ENSTRUT00000022363, which is hereby incorporated


by reference in its entirety):








127
      ATGG CCACGGGAGG GATCACAACA CTTCCATCCA CACCTGAAGA CGGCGGCAGC


181
GGCGGTTTTC CTCCCGGGAG CTTCAAGGAT CCCAAAAGGC TGTACTGTAA AAACGGCGGC


241
TTCTTCCTGA GGATCAGGTC CGACGGGGCC GTGGACGGAA CCCGGGAGAA GACTGACCCC


301
CACATAAAGC TTCAGCTCCA GGCGACCTCT GTGGGGGAGG TGGTCATCAA GGGGGTTTGT


361
GCTAATCGTT ATCTGGCCAT GAACAGAGAT GGACGACTGT TTGGAATGAA ACGAGCGACG


421
GATGAATGCC ACTTCTTAGA GCGGCTCGAG AGCAACAACT ACAACACCTA CCGCTCCAGG


481
AAGTACCCCA ACATGTTTGT GGGACTGACG CGAACTGGCA ACTACAAGTC TGGGACTAAA


541
ACTGGACCGG GCCAAAAGGC CATCCTCTTT CTTCCGATGT CGGCCAAATA CTAA


 







Rainbow trout FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 216)


(GenBank accession no. NM_001124536, which is hereby incorporated by


reference in its entirety):








390
                               A TGGCCACAGG AGAAATCACC ACTCTACCCG


421
CCACACCTGA AGATGGAGGC AGTGGCGGCT TCCTTCCAGG AAACTTTAAG GAGCCCAAGA


481
GGTTGTACTG TAAAAATGGA GGCTACTTCT TGAGGATAAA CTCTAACGGA AGCGTGGACG


541
GGATCAGAGA TAAGAACGAC CCCCACAATA AGCTTCAACT CCAGGCGACC TCAGTGGGGG


601
AAGTAGTAAT CAAAGGGGTC TCAGCCAACC GCTATCTGGC CATGAATGCA GATGGAAGAC


661
TGTTTGGACC GAGACGGACA ACAGATGAAT GCTACTTCAT GGAGAGGCTG GAGAGTAACA


721
ACTACAACAC CTACCGCTCT CGAAAGTACC CTGAAATGTA TGTGGCACTG AAAAGGACTG


781
GCCAGTACAA GTCAGGATCC AAAACTGGAC CCGGCCAAAA AGCCATCCTC TTCCTCCCCA


841
TGTCAGCCAG ACGCTGA


 







Salmon FGF2 gene coding sequence (1-150) (SEQ ID NO: 217) (GenBank


accession no. EU816603, which is hereby incorporated by reference in its


entirety):








99402
                                             ATGGCCACA GGAGAAATCA


99421
CCACTCTACC CGCCACACCT GAAGATGGAG GCAGTGGCGG CTTCCCTCCA GGAAACTTTA


99481
AGGATCCCAA GAGGCTGTAC TGTAAAAACG GGGGCTACTT CTTGAGAATA AACTCTAATG


99541
GAAGCGTGGA CGGGATCCGA GAGAAGAACG ACCCCCACA


100968
                                                   AAC AGCCTCAATT


100981
TGTCAGGGCA TGGACTCTTC AAGGTGTCAA ACGTTCCACA GGGATGCTGG CCCATGTTGA


101041
CTCCAACGCT TCCCACAATT GTGTCAAGGT GGCTGGATGT TCTTTGGGAG


101845
                          AATTTG GCAGTATGTC CAACCGGCCT CATAACCGCA


101881
GACCACGTGT AGCTACACCA GCCCAGGACC TCCACATCCG GCTTCTTCAT CTACGGGATC


101941
GTCTGAAACC AGCCACCCGA ACAGCTGATA AAACTGAGGA GTATTTCTGT CTGTAA


 







Zebrafish FGF2 gene coding sequence (aa 1-154) (SEQ ID NO: 218) (GenBank


accession no. AY269790, which is hereby incorporated by reference in its


entirety):








43
                                              ATGGCCAC CGGAGGGATC


61
ACCACACTCC CGGCCGCTCC GGACGCCGAA AACAGCAGCT TTCCCGCGGG CAGCTTCAGG


121
GATCCCAAGC GCCTGTACTG CAAAAACGGA GGATTCTTCC TGCGGATCAA CGCGGACGGC


181
CGAGTGGACG GAGCCCGAGA CAAGAGCGAC CCGCACATTC GTCTGCAGCT GCAGGCGACG


241
GCAGTGGGTG AAGTACTCAT TAAAGGCATC TGTACCAACC GTTTCCTTGC CATGAACGCA


301
GACGGACGAC TGTTCGGGAC GAAAAGGACC ACAGATGAAT GTTATTTCCT GGAGCGCCTG


361
GAGTCCAACA ACTACAACAC ATACAGATCC CGCAAGTATC CCGACTGGTA CGTGGCTCTG


421
AAGAGAACCG GCCAGTATAA AAGCGGCTCT AAAACCAGCC CGGGACAGAA GGCCATCCTG


481
TTTCTGCCCA TGTCGGCCAA ATGCTGA


 







Nile tilapia FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 219)


(GenBank accession no. XM_003443364, which is hereby incorporated by


reference in its entirety):








1
ATGGCCACGG GAGGAATCAC AACACTTCCC GCTACACCTG AAGACGGCGG CAGCAGCGGC


61
TTTCCTCCTG GGAACTTCAA GGACCCTAAA AGGCTGTACT GTAAAAATGG TGGCTTCTTC


121
TTGAGGATAA AATCTGATGG AGGAGTGGAT GGAATACGAG AGAAAAACGA CCCCCACATA


181
AAGCTTCAAC TCCAGGCGAC CTCAGTGGGA GAAGTGGTCA TCAAAGGGAT TTGTGCAAAC


241
CGATATCTGG CAATGAACAG AGATGGACGA CTGTTTGGAG CGAGAAGAGC AACAGATGAG


301
TGCTACTTCT TAGAGCGGCT CGAGAGCAAC AACTACAACA CCTACCGCTC CAGGAAGTAC


361
CCAAACATGT ACGTGGCGCT GAAGCGGACT GGCCAGTACA AGTCTGGAAG CAAAACTGGA


421
CCGGGTCAAA AGGCAATTCT CTTTCTCCCA ATGTCTGCTA AATGCTAA


 







Medaka FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 220) (Ensembl


accession no. ENSORLT00000025835, which is hereby incorporated by


reference in its entirety):








1
ATGGCTACGG GAGAAATCAC AACACTTCCC TCCCCAGCTG AAAACAGCAG AAGCGATGGC


61
TTTCCTCCAG GGAACTACAA GGATCCTAAG AGGCTCTACT GTAAAAATGG AGGTTTGTTT


121
TTGAGGATTA AACCTGATGG AGGAGTGGAT GGAATCCGGG AAAAAAAAGA TCCCCACGTT


181
AAGCTTCGCC TTCAGGCTAC CTCAGCGGGA GAGGTGGTGA TCAAAGGAGT TTGTTCAAAC


241
AGATATCTGG CGATGCATGG AGATGGACGT CTATTTGGAG TGAGACAAGC AACAGAGGAA


301
TGCTACTTCT TGGAGCGACT AGAGAGCAAC AACTATAACA CCTATCGCTC TAAAAAGTAC


361
CCAAACATGT ACGTGGCACT GAAGCGGACA GGCCAGTACA AACCTGGAAA CAAAACTGGA


421
CCAGGTCAAA AGGCCATTCT CTTTCTGCCT ATGTCTGCCA AGTACTAA









As noted above, also encompassed within the present invention are portions of paracrine FGFs other than FGF1 and/or FGF2 (e.g., FGF4, FGF5, FGF6, FGF9, FGF16, and FGF20). The portion of the paracrine FGF may be from human FGF4, FGF5, FGF6, FGF9, FGF16, and/or FGF20 having the amino acid sequences shown in Table 5, or orthologs thereof







TABLE 5







Amino acid sequence of human FGF4(SEQ ID NO: 221)


(GenBank accession no. NP_001998,


which is hereby incorporated by reference in its entirety):








1
MSGPGTAAVA LLPAVLLALL APWAGRGGAA APTAPNGTLE AELERRWESL VALSLARLPV


61
AAQPKEAAVQ SGAGDYLLGI KRLRRLYCNV GIGFHLQALP DGRIGGAHAD TRDSLLELSP


121
VERGVVSIFG VASRFFVAMS SKGKLYGSPF FTDECTFKEI LLPNNYNAYE SYKYPGMFIA


181
LSKNGKTKKG NRVSPTMKVT HFLPRL


 







Amino acid sequence of human FGF5(SEQ ID NO: 222)


(GenBank Accession No. NP_004455,


which is hereby incorporated by reference in its entirety):








1
MSLSFLLLLF FSHLILSAWA HGEKRLAPKG QPGPAATDRN PRGSSSRQSS SSAMSSSSAS


61
SSPAASLGSQ GSGLEQSSFQ WSPSGRRTGS LYCRVGIGFH LQIYPDGKVN GSHEANMLSV


121
LEIFAVSQGI VGIRGVFSNK FLAMSKKGKL HASAKFTDDC KFRERFQENS YNTYASAIHR


181
TEKTGREWYV ALNKRGKAKR GCSPRVKPQH ISTHFLPRFK QSEQPELSFT VTVPEKKKPP


241
SPIKPKIPLS APRKNTNSVK YRLKFRFG


 







Amino acid sequence of human FGF6(SEQ ID NO: 223)


(NP_066276,


which is hereby incorporated by reference in its entirety):








1
MALGQKLFIT MSRGAGRLQG TLWALVFLGI LVGMVVPSPA GTRANNTLLD SRGWGTLLSR


61
SRAGLAGEIA GVNWESGYLV GIKRQRRLYC NVGIGFHLQV LPDGRISGTH EENPYSLLEI


121
STVERGVVSL FGVRSALFVA MNSKGRLYAT PSFQEECKFR ETLLPNNYNA YESDLYQGTY


181
IALSKYGRVK RGSKVSPIMT VTHFLPRI


 







Amino acid sequence of human FGF9(SEQ ID NO: 224)


(GenBank accession no. NP_002001,


which is hereby incorporated by reference in its entirety):








1
MAPLGEVGNY FGVQDAVPFG NVPVLPVDSP VLLSDHLGQS EAGGLPRGPA VTDLDHLKGI


61
LRRRQLYCRT GFHLEIFPNG TIQGTRKDHS RFGILEFISI AVGLVSIRGV DSGLYLGMNE


121
KGELYGSEKL TQECVFREQF EENWYNTYSS NLYKHVDTGR RYYVALNKDG TPREGTRTKR


181
HQKFTHFLPR PVDPDKVPEL YKDILSQS


 







Amino acid sequence of human FGF16(SEQ ID NO: 225)


(GenBank accession no. NP_003859,


which is hereby incorporated by reference in its entirety):








1
MAEVGGVFAS LDWDLHGFSS SLGNVPLADS PGFLNERLGQ IEGKLQRGSP TDFAHLKGIL


61
RRRQLYCRTG FHLEIFPNGT VHGTRHDHSR FGILEFISLA VGLISIRGVD SGLYLGMNER


121
GELYGSKKLT RECVFREQFE ENWYNTYAST LYKHSDSERQ YYVALNKDGS PREGYRTKRH


181
QKFTHFLPRP VDPSKLPSMS RDLFHYR


 







Amino acid sequence of human FGF20(SEQ ID NO: 226)


(GenBank accession no. NP_062825,


which is hereby incorporated by reference in it entirety):








1
MAPLAEVGGF LGGLEGLGQQ VGSHFLLPPA GERPPLLGER RSAAERSARG GPGAAQLAHL


61
HGILRRRQLY CRTGFHLQIL PDGSVQGTRQ DHSLFGILEF ISVAVGLVSI RGVDSGLYLG


121
MNDKGELYGS EKLTSECIFR EQFEENWYNT YSSNIYKHGD TGRRYFVALN KDGTPRDGAR


181
SKRHQKFTHF LPRPVDPERV PELYKDLLMY T









It will be understood that the portion of the paracrine FGF according to the present invention may be derived from a nucleotide sequence that encodes human FGF4, FGF5, FGF6, FGF9, FGF16, and/or FGF20 having the nucleotide sequences shown in Table 6, or orthologs thereof.







TABLE 6







Human FGF4 gene coding sequence (1-206) (SEQ ID NO: 227)


(GenBank accession no. NM_002007,


which is hereby incorporated by reference in its entirety):








320
                    A TGTCGGGGCC CGGGACGGCC GCGGTAGCGC TGCTCCCGGC


361
GGTCCTGCTG GCCTTGCTGG CGCCCTGGGC GGGCCGAGGG GGCGCCGCCG CACCCACTGC


421
ACCCAACGGC ACGCTGGAGG CCGAGCTGGA GCGCCGCTGG GAGAGCCTGG TGGCGCTCTC


481
GTTGGCGCGC CTGCCGGTGG CAGCGCAGCC CAAGGAGGCG GCCGTCCAGA GCGGCGCCGG


541
CGACTACCTG CTGGGCATCA AGCGGCTGCG GCGGCTCTAC TGCAACGTGG GCATCGGCTT


601
CCACCTCCAG GCGCTCCCCG ACGGCCGCAT CGGCGGCGCG CACGCGGACA CCCGCGACAG


661
CCTGCTGGAG CTCTCGCCCG TGGAGCGGGG CGTGGTGAGC ATCTTCGGCG TGGCCAGCCG


721
GTTCTTCGTG GCCATGAGCA GCAAGGGCAA GCTCTATGGC TCGCCCTTCT TCACCGATGA


781
GTGCACGTTC AAGGAGATTC TCCTTCCCAA CAACTACAAC GCCTACGAGT CCTACAAGTA


841
CCCCGGCATG TTCATCGCCC TGAGCAAGAA TGGGAAGACC AAGAAGGGGA ACCGAGTGTC


901
GCCCACCATG AAGGTCACCC ACTTCCTCCC CAGGCTGTGA


 







Human FGF5 gene coding sequence (1-268) (SEQ ID NO: 228)


(GenBank Accession No. NM_004464,


which is hereby incorporated by reference in its entirety):








238
                                                              ATG


241
AGCTTGTCCT TCCTCCTCCT CCTCTTCTTC AGCCACCTGA TCCTCAGCGC CTGGGCTCAC


301
GGGGAGAAGC GTCTCGCCCC CAAAGGGCAA CCCGGACCCG CTGCCACTGA TAGGAACCCT


361
AGAGGCTCCA GCAGCAGACA GAGCAGCAGT AGCGCTATGT CTTCCTCTTC TGCCTCCTCC


421
TCCCCCGCAG CTTCTCTGGG CAGCCAAGGA AGTGGCTTGG AGCAGAGCAG TTTCCAGTGG


481
AGCCCCTCGG GGCGCCGGAC CGGCAGCCTC TACTGCAGAG TGGGCATCGG TTTCCATCTG


541
CAGATCTACC CGGATGGCAA AGTCAATGGA TCCCACGAAG CCAATATGTT AAGTGTTTTG


601
GAAATATTTG CTGTGTCTCA GGGGATTGTA GGAATACGAG GAGTTTTCAG CAACAAATTT


661
TTAGCGATGT CAAAAAAAGG AAAACTCCAT GCAAGTGCCA AGTTCACAGA TGACTGCAAG


721
TTCAGGGAGC GTTTTCAAGA AAATAGCTAT AATACCTATG CCTCAGCAAT ACATAGAACT


781
GAAAAAACAG GGCGGGAGTG GTATGTGGCC CTGAATAAAA GAGGAAAAGC CAAACGAGGG


841
TGCAGCCCCC GGGTTAAACC CCAGCATATC TCTACCCATT TTCTGCCAAG ATTCAAGCAG


901
TCGGAGCAGC CAGAACTTTC TTTCACGGTT ACTGTTCCTG AAAAGAAAAA GCCACCTAGC


961
CCTATCAAGC CAAAGATTCC CCTTTCTGCA CCTCGGAAAA ATACCAACTC AGTGAAATAC


1021
AGACTCAAGT TTCGCTTTGG ATAA


 







Human FGF6 gene coding sequence (1-208) (SEQ ID NO: 229)


(NM_020996,


which is hereby incorporated by reference in its entirety):








45
                                                ATGGCC CTGGGACAGA


61
AACTGTTCAT CACTATGTCC CGGGGAGCAG GACGTCTGCA GGGCACGCTG TGGGCTCTCG


121
TCTTCCTAGG CATCCTAGTG GGCATGGTGG TGCCCTCGCC TGCAGGCACC CGTGCCAACA


181
ACACGCTGCT GGACTCGAGG GGCTGGGGCA CCCTGCTGTC CAGGTCTCGC GCGGGGCTAG


241
CTGGAGAGAT TGCCGGGGTG AACTGGGAAA GTGGCTATTT GGTGGGGATC AAGCGGCAGC


301
GGAGGCTCTA CTGCAACGTG GGCATCGGCT TTCACCTCCA GGTGCTCCCC GACGGCCGGA


361
TCAGCGGGAC CCACGAGGAG AACCCCTACA GCCTGCTGGA AATTTCCACT GTGGAGCGAG


421
GCGTGGTGAG TCTCTTTGGA GTGAGAAGTG CCCTCTTCGT TGCCATGAAC AGTAAAGGAA


481
GATTGTACGC AACGCCCAGC TTCCAAGAAG AATGCAAGTT CAGAGAAACC CTCCTGCCCA


541
ACAATTACAA TGCCTACGAG TCAGACTTGT ACCAAGGGAC CTACATTGCC CTGAGCAAAT


601
ACGGACGGGT AAAGCGGGGC AGCAAGGTGT CCCCGATCAT GACTGTCACT CATTTCCTTC


661
CCAGGATCTA A


 







Human FGF9 gene coding sequence (1-208)(SEQ ID NO: 230)


(GenBank accession no. NM_002010,


which is hereby incorporated by reference in its entirety):








838
                                                              ATG


841
GCTCCCTTAG GTGAAGTTGG GAACTATTTC GGTGTGCAGG ATGCGGTACC GTTTGGGAAT


901
GTGCCCGTGT TGCCGGTGGA CAGCCCGGTT TTGTTAAGTG ACCACCTGGG TCAGTCCGAA


961
GCAGGGGGGC TCCCCAGGGG ACCCGCAGTC ACGGACTTGG ATCATTTAAA GGGGATTCTC


1021
AGGCGGAGGC AGCTATACTG CAGGACTGGA TTTCACTTAG AAATCTTCCC CAATGGTACT


1081
ATCCAGGGAA CCAGGAAAGA CCACAGCCGA TTTGGCATTC TGGAATTTAT CAGTATAGCA


1141
GTGGGCCTGG TCAGCATTCG AGGCGTGGAC AGTGGACTCT ACCTCGGGAT GAATGAGAAG


1201
GGGGAGCTGT ATGGATCAGA AAAACTAACC CAAGAGTGTG TATTCAGAGA ACAGTTCGAA


1261
GAAAACTGGT ATAATACGTA CTCATCAAAC CTATATAAGC ACGTGGACAC TGGAAGGCGA


1321
TACTATGTTG CATTAAATAA AGATGGGACC CCGAGAGAAG GGACTAGGAC TAAACGGCAC


1381
CAGAAATTCA CACATTTTTT ACCTAGACCA GTGGACCCCG ACAAAGTACC TGAACTGTAT


1441
AAGGATATTC TAAGCCAAAG TTGA


 







Human FGF16 gene coding sequence (1-207) (SEQ ID NO: 231)


(GenBank accession no. NM_003868,


which is hereby incorporated by reference in its entirety):








1
ATGGCAGAGG TGGGGGGCGT CTTCGCCTCC TTGGACTGGG ATCTACACGG CTTCTCCTCG


61
TCTCTGGGGA ACGTGCCCTT AGCTGACTCC CCAGGTTTCC TGAACGAGCG CCTGGGCCAA


121
ATCGAGGGGA AGCTGCAGCG TGGCTCACCC ACAGACTTCG CCCACCTGAA GGGGATCCTG


181
CGGCGCCGCC AGCTCTACTG CCGCACCGGC TTCCACCTGG AGATCTTCCC CAACGGCACG


241
GTGCACGGGA CCCGCCACGA CCACAGCCGC TTCGGAATCC TGGAGTTTAT CAGCCTGGCT


301
GTGGGGCTGA TCAGCATCCG GGGAGTGGAC TCTGGCCTGT ACCTAGGAAT GAATGAGCGA


361
GGAGAACTCT ATGGGTCGAA GAAACTCACA CGTGAATGTG TTTTCCGGGA ACAGTTTGAA


421
GAAAACTGGT ACAACACCTA TGCCTCAACC TTGTACAAAC ATTCGGACTC AGAGAGACAG


481
TATTACGTGG CCCTGAACAA AGATGGCTCA CCCCGGGAGG GATACAGGAC TAAACGACAC


541
CAGAAATTCA CTCACTTTTT ACCCAGGCCT GTAGATCCTT CTAAGTTGCC CTCCATGTCC


601
AGAGACCTCT TTCACTATAG GTAA


 







Human FGF20 gene coding sequence (1-211) (SEQ ID NO: 232)


(GenBank accession no. NM_019851,


which is hereby incorporated by reference in its entirety):








134
              ATGGCTC CCTTAGCCGA AGTCGGGGGC TTTCTGGGCG GCCTGGAGGG


181
CTTGGGCCAG CAGGTGGGTT CGCATTTCCT GTTGCCTCCT GCCGGGGAGC GGCCGCCGCT


241
GCTGGGCGAG CGCAGGAGCG CGGCGGAGCG GAGCGCGCGC GGCGGGCCGG GGGCTGCGCA


301
GCTGGCGCAC CTGCACGGCA TCCTGCGCCG CCGGCAGCTC TATTGCCGCA CCGGCTTCCA


361
CCTGCAGATC CTGCCCGACG GCAGCGTGCA GGGCACCCGG CAGGACCACA GCCTCTTCGG


421
TATCTTGGAA TTCATCAGTG TGGCAGTGGG ACTGGTCAGT ATTAGAGGTG TGGACAGTGG


481
TCTCTATCTT GGAATGAATG ACAAAGGAGA ACTCTATGGA TCAGAGAAAC TTACTTCCGA


541
ATGCATCTTT AGGGAGCAGT TTGAAGAGAA CTGGTATAAC ACCTATTCAT CTAACATATA


601
TAAACATGGA GACACTGGCC GCAGGTATTT TGTGGCACTT AACAAAGACG GAACTCCAAG


661
AGATGGCGCC AGGTCCAAGA GGCATCAGAA ATTTACACAT TTCTTACCTA GACCAGTGGA


721
TCCAGAAAGA GTTCCAGAAT TGTACAAGGA CCTACTGATG TACACTTGA









As noted above, the chimeric protein includes a portion of a paracrine FGF coupled to a C-terminal region derived from an FGF23. FGF23 is an endocrine FGF that was cloned by Itoh et al. at Kyoto University (WO 01/66596 to Itoh et al., which is hereby incorporated by reference in its entirety). FGF23 mRNA is expressed mainly in the brain, preferentially in the ventrolateral thalamic nucleus. It is also expressed in the thymus at low levels (Yamashita et al., “Identification of a Novel Fibroblast Growth Factor, FGF-23, Preferentially Expressed in the Ventrolateral Thalamic Nucleus of the Brain,” Biochem Biophys Res Comm 277(2):494-498 (2000), which is hereby incorporated by reference in its entirety). The tissue with the highest level of FGF23 expression is bone (osteocytes and osteoblasts), where it is highly expressed during phases of active bone remodeling (Riminucci et al., “FGF-23 in Fibrous Dysplasia of Bone and its Relationship to Renal Phosphate Wasting,” J Clin Invest 112:683-692 (2003), which is hereby incorporated by reference in its entirety). Expression of FGF23 in dendritic cells has also been reported (Katoh et al., “Comparative Genomics on Mammalian Fgf6-Fgf23 Locus.,” Int J Mol Med 16(2):355-358 (2005), which is hereby incorporated by reference in its entirety). See also Zhang et al., “Receptor Specificity of the Fibroblast Growth Factor Family,” J Biol Chem 281(23):15694-15700; Yu et al., “Analysis of the Biochemical Mechanisms for the Endocrine Actions of Fibroblast Growth Factor-23,” Endocrinology 146(11):4647-4656, which are hereby incorporated by reference in their entirety.
In one embodiment the C-terminal region of the FGF23 molecule includes an α-Klotho-FGFR complex binding domain. In one embodiment, the C-terminal region is from human FGF23 having the amino acid sequence of SEQ ID NO: 233 (GenBank accession no. AAG09917, which is hereby incorporated by reference in its entirety), as follows:









1
MLGARLRLWV CALCSVCSMS VLRAYPNASP LLGSSWGGLI HLYTATARNS YHLQIHKNGH



 


61
VDGAPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHYFDP ENCRFQHQTL


 


121
ENGYDVYHSP QYHFLVSLGR AKRAFLPGMN PPPYSQFLSR RNEIPLIHFN TPIPRRHTRS


 


181
AEDDSERDPL NVLKPRARMT PAPASCSQEL PSAEDNSPMA SDPLGVVRGG RVNTHAGGTG


 


241
PEGCRPFAKF I






In one embodiment, the C-terminal portion from FGF23 of the chimeric protein according to the present invention includes an amino acid sequence beginning at any of amino acid residues 161-180 and ending at any of amino acid residues 200-251 of SEQ ID NO: 233. In one embodiment, the C-terminal portion from FGF23 of the chimeric protein according to the present invention comprises amino acid residues 161-200, 162-200, 163-200, 164-200, 165-200, 166-200, 167-200, 168-200, 169-200, 170-200, 171-200, 172-200, 173-200, 174-200, 175-200, 176-200, 177-200, 178-200, 179-200, 180-200, 161-205, 162-205, 163-205, 164-205, 165-205, 166-205, 167-205, 168-205, 169-205, 170-205, 171-205, 172-205, 173-205, 174-205, 175-205, 176-205, 177-205, 178-205, 179-205, 180-205, 161-251, 162-251, 163-251, 164-251, 165-251, 166-251, 167-251, 168-251, 169-251, 170-251, 171-251, 172-251, 173-251, 174-251, 175-251, 176-251, 177-251, 178-251, 179-251, or 180-251 of SEQ ID NO: 233.
In one embodiment, the C-terminal portion from FGF23 of the chimeric protein according to the present invention includes one or more deletions or substitutions while retaining the ability to bind the binary α-Klotho-FGFR complex. In one embodiment, the C-terminal portion from FGF23 of the chimeric protein according to the present invention includes a substitution at amino acid residues (or amino acid residues corresponding to) R176 and/or R179 of SEQ ID NO:233. In one embodiment, the R176 substitution is a R176Q/W substitution and/or the R179 substitution is a R179Q/W substitution. In one embodiment, the C-terminal portion from FGF23 of the chimeric protein according to the present invention includes amino acid residues 161-200, 162-200, 163-200, 164-200, 165-200, 166-200, 167-200, 168-200, 169-200, 170-200, 171-200, 172-200, 173-200, 174-200, 175-200, 176-200, 177-200, 178-200, 179-200, 180-200, 161-205, 162-205, 163-205, 164-205, 165-205, 166-205, 167-205, 168-205, 169-205, 170-205, 171-205, 172-205, 173-205, 174-205, 175-205, 176-205, 177-205, 178-205, 179-205, 180-205, 161-251, 162-251, 163-251, 164-251, 165-251, 166-251, 167-251, 168-251, 169-251, 170-251, 171-251, 172-251, 173-251, 174-251, 175-251, 176-251, 177-251, 178-251, 179-251, or 180-251 of SEQ ID NO: 233, where one or both of R176 and R179 are substituted. In one embodiment, the R176 substitution is a R176Q/W substitution and/or the R179 substitution is a R179Q/W substitution.
In one embodiment, the FGF23 according to the present invention is from a mammal. In one embodiment, the FGF23 according to the present invention is from a vertebrate. It will be understood that this includes orthologs of human FGF23, or a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. In one embodiment of the present invention, the FGF23 according to the present invention is from Gorilla gorilla, Nomascus leucogenys, Macaca mulatta, Macaca fascicularis, Pan troglodytes, Callithrix jacchus, Loxodonta Africana, Erinaceus telfairi, Erinaceus europaeus, Otolemur garnettii, Oryctolagus cuniculus, Equus caballus, Ailuropoda melanoleuca, Ochotona princeps, Bos taurus, Sus scrofa, Canis lupus familiaris, Cavia porcellus, Cricetulus griseus, Tupaia belangeri, Rattus norvegicus, Mus musculus, Pteropus vampyrus, Myotis lucifugus, Sarcophilus harrisii, Monodelphis domestica, Dasypus novemcinctus, Macropus eugenii, Taeniopygia guttata, Gallus gallus, Meleagris gallopavo, Anolis carolinensis, Latimeria chalumnae, Xenopus silurana tropicalis, Felis catus, Pelodiscus sinensis, Mustela putorius furo, Microcebus murinus, Pongo abelii, Sorex araneus, Tetraodon nigroviridis, Oreochromis niloticus, or Danio rerio. 
In one embodiment, FGF23 according to the present invention is from a non-human FGF23 (or an FGF23 ortholog) having an amino acid sequence as shown in Table 7. The portions of an ortholog of human FGF23 of a chimeric protein according to the present invention include portions corresponding to the above-identified amino acid sequences of human FGF23. Corresponding portions may be determined by, for example, sequence analysis and structural analysis.







TABLE 7







Amino acid sequence of Gorilla gorilla (gorilla)


FGF23 (SEQ ID NO: 234)


(Ensembl accession no. ENSGGOP00000002917,


which is hereby incorporated by reference in its entirety):








1
MLGARLRLWV CALCSVCSLS VLRAYPNASP LLGSSWGGLI HLYTATARNS YHLQIHKNGH


61
VDGAPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHYFDP ENCRFQHQTL


121
ENGYDVYHSP QYHFLVSLGR AKRAFLPGMN PPPYSQFLSR RNEIPLIHFN TPIPRRHTRS


181
AEDDSERDPL NVLKPRARMT PAPASCSQEL PSAEDNSPMA SDPLGVVRGG RVNTYAGGTG


241
PEGCRPFPKF I


 







Amino acid sequence of Nomascus leucogenys


(Northern white-cheeked gibbon) FGF23(SEQ ID


NO: 235) (GenBank accession no. XP_003273749,


which is hereby incorporated by reference in its entirety):








1
MLGARLRLWV CALCSVCSMS VLRAYPNASP LLGSSWGGLI HLYTATARNS YHLQIHKNGH


61
VDGAPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHYFNP ENCRFQHQTL


121
ENGYDVYHSP QHHFLVSLGR AKRAFLPGMN PPPYSQFLSR RNEIPLLHFN TPTPRRHTRS


181
AEDDSERDPL NVLKPRARMT PAPASCSQEL LSSEDNSPMA SDPLGVVRGG RVNTHAGGTG


241
PEGCRPFPKF I


 







Amino acid sequence of Macaca mulatta (rhesus monkey)


FGF23 (SEQ ID NO: 236) (GenBank accession


no. NP_001181066,


which is hereby incorporated by reference in its entirety):








1
MLGARLRLWV CALCSVCSMS VIRAYPNASP LLGSSWGGLI HLYTATARNS YHLQIHKNGH


61
VDGAPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHYFNP ENCRFRHWTL


121
ENGYDVYHSP QHHFLVSLGR AKRAFLPGMN PPPYSQFLSR RNEIPLIHFN TPRPRRHTRS


181
AEDDSERDPL NVLKPRARMT PAPASCSQEL PSAEDNSPVA SDPLGVVRGG RVNTHAGGTG


241
PEACRPFPKF I


 







Amino acid sequence of Macaca fascicularis


(crab-eating macaque) FGF23 (SEQ ID NO:


237) (GenBank accession no. EHH66001,


which is hereby incorporated by reference in its entirety):








1
MLGARLRLWV CALCSVCSMS VIRAYPNASP LLGSSWGGLI HLYTATARNS YHLQIHKNGH


61
VDGAPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHYFNP ENCRFRHWTL


121
ENGYDVYHSP QHHFLVSLGR AKRAFLPGMN PPPYSQFLSR RNEIPLIHFN TPRPRRHTRS


181
AEDDSERDPL NVLKPRARMT PAPASCSQEL PSAEDNSPVA SDPLGVVRAG RVNTHAGGTG


241
PEACRPFPKF I


 







Amino acid sequence of Pan troglodytes (chimpanzee)


FGF23(SEQ ID NO: 238) (GenBank


accession no. XP_001157070,


which is hereby incorporated by reference in its entirety):








1
MLGARLRLWV CALCSVCSVS VLRAYPNASP LLGSSWGGLI HLYTATARNS YHLQIHKNGH


61
VDGAPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHYFNP ENCRFQHQTL


121
ENGYDVYYSP QYHFLVSLGR AKRAFLPSMN PPPYSQFLSR RNEIPLIHFN TPIPRRHTRS


181
AEDDSERDPL NVLKPRARMT PAPASCSQEL PSAEDNSPMA SDPLGVVRGG RVNTHAGGTG


241
PEGCRPFPKF I


 







Amino acid sequence of Callithrix jacchus


(white-tufted-ear marmoset) FGF23 (SEQ ID


NO: 239) (GenBank accession no. XP_002752281,


which is hereby incorporated by reference in its entirety):








1
MLGARLRLWV CALCSVCSMS VLRAYPNASP LLASSWGGLI HLYTATARNS YHLQIHKNGH


61
VDGAPHQTIY SALLIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHYFNP ENCRFRPQRL


121
ENGYDVYQSP QHHFLVSLGR AKRAFLPGMN PPPYSQFLSR RNEIPLIHFN TPKPRRHTRS


181
AEDDPELDPL NVLKSRVRMT PAPASCSQEL LSAEDNSPVG SDPLGMVRGG RVNSHAEGTG


241
PEGCSPFPKL I


 







Amino acid sequence of Loxodonta africana


(elephant) FGF23 (SEQ ID NO: 240)


(GenBank accession no. XP_003410677,


which is hereby incorporated by reference in its entirety):








1
MLGARLRLWV CTLCSACSMC SVRAYPNASP LLHSSWGGLT HLYTATARNS YHLQIHKDGH


61
VDGTPDQTIY SALIIRSEEA GFVVITGVMS RRYLCMDFRG NIFGSHYFNP ENCRFKHWTL


121
ENGYDVYHSP QHHFLVSLGR VKKAFLPGMN PPPYSQFLSR RNEIPLIYFN TPKPRRHTRS


181
AEDDSERDPL NVLKPRPRMT PAPASCSQEL LSAEDNSVVA NDPLGVVRSN RVNTHAGGIG


241
VERCRPFPKF I


 







Amino acid sequence of Erinaceus telfairi


(lesser hedgehog tenrec) FGF23 (SEQ ID NO:


241) (Ensembl accession no. ENSETEP00000001298,


which is hereby incorporated by reference in its entirety):








1
MLGAHLRLWV CALCSVSAMY HVRAYPNASP LLGTSWAGLT HLYTATARNS FHLQIHKDGH


61
VDGTPHQTIY SALMIRSEDS GFVVITGVMS RRYLCMDFRG NIFGSHYFTA DSCRFRQRTL


121
ENGYDVYHSP QHHFLISLGR AKRVFLPGMN PPPYSQFLSR RNEIPLIHFN TPRPRRHTRS


181
AEEEVEQDPL NVLKPRPRMT PAPASCSQEL PSAEDNSALA SDPLGVVRGK KLNTHAVGMG


241
AERCRPFPKF


 







Amino acid sequence of Erinaceus europaeus


(hedgehog) FGF23 (SEQ ID NO: 242) (Ensembl


accession no. ENSEEUP00000007211,


which is hereby incorporated by reference in its entirety):








1
MLGAHLGLVV CALVSRAYPN ASPLLGFSWG GLTHLYTATA RNSYHLQIHK DGHVDGSPQQ


61
TIY------- --AGFVMITG VMSRRYLCMD FRSNIFGSHH FAPESCRFRH RTLENGYDVY


121
HSPQHHFLVS LGRAKRAFLP GTNPPPYSQF LSRRNEVPLI HFNTPRPRRH TRSAEDNSEL


181
DPLNVLKPRP RMTPAPASCS QELPSAEDNS MVASDPLGVV RANRVNTHAG GLGVDKCRPF


241
PKFI


 







Amino acid sequence of Otolemur garnettii


(bushbaby) FGF23 (SEQ ID NO: 243) (Ensembl


accession no. ENSOGAP00000004657,


which is hereby incorporated by reference in its entirety):








1
MLGTCLRLWV CALCSVCSVS IVRAYPNASP LLSSSWGGLT HLYTASARNS YHLQIHKDGH


61
VDGTPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFKG NIFGSHSFHP ESCRFRHRTL


121
ENGYDVYLSP QHHFLVSLGR SKRPFLPGMN PPPFSQFLSR RNDIPLIHFN TPRPRRHTRS


181
AEDNDSELDP LNVLKPRPRA TPGPASCSQE LPSAEDNSLV ASDPLGVVRG NRVNAHAGRA


241
GLDRCRPFPR YF


 







Amino acid sequence of Oryctolagus cuniculus


(rabbit) FGF23 (SEQ ID


NO: 244) (GenBank accession no. XP_002712872,


which is hereby incorporated by reference in its entirety):








1
MLGARLLRLL VCALGSVCSW CVVRAYPDTS PLLSSSWAGL THLYTATARN SYHLQIHKDG


61
QVDGTPHQTI YSALMIRSED AGFVVITGVM SRRYLCMDFR GNIFGSHYFD PQNCRFRHRT


121
LENGYDVYHS PEHHFLVSLG RAKRPFLPGM NPPPYSQFLS RRNEIPLIHF NTPRPRRHTR


181
SAEDAWEQDP LNVLKPRFRL TPAPASCSQE APSAEDNGLV ASDPFGVLRG NRVNMHGDRM


241
GPERCHHFPK FI


 







Amino acid sequence of Equus caballus


(horse) FGF23 (SEQ ID NO: 245) (GenBank


accession no. XP_001491469,


which is hereby incorporated by reference in its entirety):








1
MSGPCLGLLV YVLCSAVKAY PNASPLLDSS WGSLTHLYTA TARNSYHLQI HKDGHVDGTP


61
HQTIYSALMI RSEDAGFVVI TGVMSRRYLC MDFRGNIFGS HHFSPESCSF RQRTLENGYD


121
VYHSPQHRFL VSLGRAKRAF LPGTNPPPYS QFLSRRNEIP LVHFNTPRPR RHTRSAEDNS


181
ERDPLNVLKP RPRMTPAPAS CSQELPSAED NSVLASDPLG VVRGNRVNTH AGGAGVERCR


241
PFPKFF


 







Amino acid sequence of Ailuropoda melanoleuca


(giant panda) FGF23 (SEQ ID NO: 246)


(GenBank accession no. XP_002920496,


which is hereby incorporated by reference in its entirety):








1
MSGTRLGLLV SVLCWVGRAY PNTSPLLGSS WGGLTHLYTA SARNSYHLQI HKDGHVDGTP


61
HQTIYSALMI RSEDAGFVVI TGVMSRRYLC MDLRGNIFGS HLFSPESCRF RQRTLENGYD


121
VYHSPQHRFL VSLGQAKRTF LPGTNPPPYS QFLSRRNEIP LIHFNTPRPR RHTRSAEDTE


181
RDPLNVLKPR PRMTPAPASC SQELPSAEDN SVVASDPLGV LRGNRVNAHA GGMGVDRCRP


241
FPKFI


 







Amino acid sequence of Ochotona princeps


(pika) FGF23 (SEQ ID NO: 247)


(Ensembl accession no. ENSOPRP00000006546,


which is hereby incorporated by reference in its entirety):








1
MLGGLGLWVC VLGSVCSWRG VRAYPDTSPL LGSSWTGLTH LYTATARNSF HLQIHKDGHV


61
DGTPQQTIYS ALMIRSEDAG FVVITGVMSR RYLCMDFRGN IFGSHYFEPQ NCRFQQRTLE


121
NGYDIYHSPQ HDFLVSLGRA KRPFLPGMNP PPYSQFLSRR NEIPLILFNT PRPRRHTRSA


181
EEGWERDPLN VLKSRPRMTP APASCSREAP SAEDDGLLAS DPMGVLRGHR VDVHGGGTGR


241
DRCRPFPRFI


 







Amino acid sequence of Bos taurus (cattle)


FGF23 (SEQ ID NO: 248) (GenBank


accession no. XP_002687926,


which is hereby incorporated by reference in its entirety):








1
MLGARLGLWV CTLSCVVQAY PNSSPLLGSS WGGLTHLYTA TARNSYHLQI HGDGHVDGSP


61
QQTVYSALMI RSEDAGFVVI TGVMSRRYLC MDFTGNIFGS HHFSPESCRF RQRTLENGYD


121
VYHSPQHRFL VSLGRAKRAF LPGTNPPPYA QFLSRRNEIP LPHFAATARP RRHTRSAHDS


181
GDPLSVLKPR ARATPVPAAC SQELPSAEDS GPAASDPLGV LRGHRLDVRA GSAGAERCRP


241
FPGFA


 







Amino acid sequence of Sus scrofa (pig)


FGF23 (SEQ ID NO: 249) (GenBank


accession no. XP_001926560,


which is hereby incorporated by reference in its entirety):








1
MLGARLGLWV CTLCCAARAY PDTSPLLSSG WGGLTHLYTA TARNSYHLQI HKDGHVDGSP


61
QQTIYSALMI RSEDAGFVVI TGVMSRRYLC MDLRGNIFGS LHFSPESCRF RQRTLENGYD


121
VYHSPHYRFL VSLGRAKRAF LPGTNPPPYA QFLSRRNEIP LLHFATARPR RHTRSAHDGG


181
DPLSVLKPRA RATPAPVSCS RELPSAEDGG PAASDPLGVL RGQRLDARAG VGGAERCRPF


241
PSFA


 







Amino acid sequence of Canis lupus


familiaris (dog) FGF23 (SEQ ID NO: 250) (GenBank


accession no. XP_854580,


which is hereby incorporated by reference in its entirety):








1
MWTVEFFLFD VTGPPFKSLR EKRRESSLGL SRKIPTKKRR KRPVRHSRGI KEAVSGFKLQ


61
PAIQRAVMSG TRLGFLVSVL CWVVRAYSNT SPLLGSSWGS LTHLYTATAR NSYHLQIHKD


121
GHVDGTPHQT IYSALMIRSE DAGFVVITGV MSRRYLCMDF RGNIFGSHLF SPESCRFRQR


181
TLENGYDVYH SPQHRFLVSL GQAKRAFLPG TNPPPYSQFL SRRNEIPLVH FHTPRPRRHT


241
RSAEAPERDP LNVLKPRPRL APAPASCSQE LPSAEDPGAP ASDPLGVLRG HRANARAGGV


301
GVDRCRAFPT PI


 







Amino acid sequence of Cavia porcellus


(domestic guinea pig) FGF23 (SEQ ID NO: 251)


(GenBank accession no. XP_003463346,


which is hereby incorporated by reference in its entirety):








1
MLGTCLGLLA CTVSLVGAYP DASPLLTSSW GGLIHLYTAT ARNSYHLQIH KDGHIDGAPY


61
PTIYSALMIR SEDAGFVVIT GVTSRRFLCM DFRGNIFGSH HFNPQDCRFQ HRTLENGYDV


121
YLSPEHHFLI SLGRTKKFFL PGTNPPPYSQ FLSRRNELPL ARFVTPGPRR HTRSAEEDQG


181
RDPLSVLKLR PRATPAPASC SQELPSAEDA AQASDPLGVL RGARVHAHGG PRPARCRPGP


241
GAK


 







Amino acid sequence of Cricetulus griseus


(Chinese hamster) FGF23 (SEQ ID NO: 252)


(GenBank accession no. XP_003496132,


which is hereby incorporated by reference in its entirety):








1
MLGTCLRLLV GVLCSACSLG TVRAYPDTSP LLGSNWGSLT HLYTATARNS YHLQIHKDGR


61
VDGTPHQTIY SALMIRSEDA GFVIITGAVT RRFLCMDLRG NIFGSHHFSP ENCRFRQRTL


121
ENGYDVYLSP QHHYLVSLGR AKRPFEPGTN PPPFSQFLAR RNEVPLLRFH TARPRRHTRS


181
AEDPPEWDPL NVLKPRPRAT PVPVSCSREL PSAEEGDLAA SDPLGVLRRG RGDARGGAGG


241
VDRCRPFPRF A


 







Amino acid sequence of Tupaia belangeri


(tree shrew) FGF23 (SEQ ID NO: 253)


(Ensembl accession no. ENSTBEP00000014220,


which is hereby incorporated by reference in its entirety):








1
ALLIRPEEAG FAVITGVMSR RYLCMDFRGN IFGSHLFSPE SCRFRQRALE NGYDVYHHPQ


61
HHFLVSLGRP KRAFVPGTNP PPYSQFLARK NEIPLIHFNT PKPRRHTRSA EDNSGRDPLN


121
VLKPRPRMTP APASCSQELP SAEDNSVVAS DPLGVLRGNR VNTHAGGWGV DRCRPFPRFI


 







Amino acid sequence of Rattus norvegicus


(Norway rat) FGF23 (SEQ ID NO: 254)


(GenBank accession no. NP_570110,


which is hereby incorporated by reference in its entirety):








1
MLGACLRLLV GALCTVCSLG TARAYSDTSP LLGSNWGSLT HLYTATARNS YHLQIHRDGH


61
VDGTPHQTIY SALMITSEDA GSVVIIGAMT RRFLCMDLRG NIFGSYHFSP ENCRFRQWTL


121
ENGYDVYLSP KHHYLVSLGR SKRIFQPGTN PPPFSQFLAR RNEVPLLHFY TARPRRHTRS


181
AEDPPERDPL NVLKPRPRAT PIPVSCSREL PSAEEGGPAA SDPLGVLRRG RGDARRGAGG


241
TDRCRPFPRF V


 







Amino acid sequence of Mus musculus


(house mouse) FGF23 (SEQ ID NO: 255)


(GenBank accession no. AAI20606,


which is hereby incorporated by reference in its entirety):








1
MLGTCLRLLV GALCTVCSLG TARAYPDTSP LLGSNWGSLT HLYTATARTS YHLQIHRDGH


61
VDGTPHQTIY SALMITSEDA GSVVITGAMT RRFLCMDLHG NIFGSLHFSP ENCKFRQWTL


121
ENGYDVYLSQ KHHYLVSLGR AKRIFQPGTN PPPFSQFLAR RNEVPLLHFY TVRPRRHTRS


181
AEDPPERDPL NVLKPRPRAT PVPVSCSREL PSAEEGGPAA SDPLGVLRRG RGDARGGAGG


241
ADRCRPFPRF V


 







Amino acid sequence of Pteropus vampyrus


(megabat) FGF23 (SEQ ID NO: 256)


(Ensembl accession no. ENSPVAP00000000222,


which is hereby incorporated by reference in its entirety):








1
MPRGSLGLLV CILCCRAYPD ASPLLSSSLG GLIHLYTATA RNGYHLQIHK DGHVDGTPHQ


61
TIYSALMIRS EDSGFVVIIG VMSRRYLCMD FKGNIFGSHH FSPESCKFRQ RTLENGYDVY


121
HSPQHHFFVS LGRAKRAFLP GTNPPPYSQF LSRRNEIPLF QFNTPRPRRH TRSVEDYKDY


181
DLDPDPLKVL RPRPRWVPAL PSCSQELPSA EDNSVVANDP LGVLRPSRVN IYRERMGKGR


241
CRPHPEFV


 







Amino acid sequence of Myotis lucifugus


(microbat) FGF23 (SEQ ID NO: 257)


(Ensembl accession no. ENSMLUP00000017312,


which is hereby incorporated by reference in its entirety):








1
MPGARLGLLV CVLALRCVVR AYPNASPLLG SSWGGLTHLY TASARNSYHL QIHKDGHVDG


61
TPHQTIYSAL MIRSEDAGFV VITGVMSRRY LCMDFRGNIF GSLFFSPSNF SFLEWKKESG


121
MDHWISRQTH FLVSPGPSQE GLPAGHNPPP YSQFLSRNEI PLFHFNTPAP RRHTRSAEEN


181
SAADPLVVLK PVPRLTPPPA SCSRELSSAE DNSVAAHDPL GVLRSSNRVN SHAPPPGPPR


241
TRQGMLLV


 







Amino acid sequence of Sarcophilus harrisii


(Tasmanian devil) FGF23 (SEQ ID NO: 258)


(Ensembl accession no. ENSSHAP00000010151,


which is hereby incorporated by reference in its entirety):








1
MSGGCLRLLF CALCSLRAIQ AFPNASPLLS LGWGGLTHLY TATARNSYHL QIHKDGHVDG


61
SPHQTIYSAL MIRSEDAGLV IITGVMSRRY LCMDIRGNIF GSHFFSPDNC RFKHRTLENG


121
YDIYHSPQNN FLISLGKAKR AFLPGMNPPP YSQFLSRRNE IPIIHFNTPE PHRHTRSAEN


181
SPDLDPMNVL KLRPRITPCS QELHSAEENS VVDDDPLEVL RNSNRLKPYP GRMSLERCLH


241
VPKAA


 







Amino acid sequence of Monodelphis domestica


(gray short-tailed opossum) FGF23 (SEQ ID NO:


259) (GenBank accession no. XP_001372436,


which is hereby incorporated by reference in its entirety):








1
MANCREKELE MYICALMIRS EDAGLVIITG VMSRRYLCMD IRGNIFGSHF FNPDNCKFKH


61
RTLENGYDIY HSPQNNFLIS LGKAKRAFLP GMNPPPYSQF LSRKNEIPII HFNTPEPHRH


121
TRSAENSPDL DPMNVLKPRP RMTPCSQELY SAEENSVVDD DPLEVLRNSN RLKPFPGRLG


181
LERCHHVPKT D


 







Amino acid sequence of Dasypus novemcinctus


(armadillo) FGF23 (SEQ ID NO: 260) (Ensembl


accession no. ENSDNOP00000004491,


which is hereby incorporated by reference in its entirety):








1
ALMISSEDAG FVVITGVMSR RYLCMDFRGN IFGSHDFTPD SCRFRQRTLE NGYDVYHSPQ


61
HHFLVSLGRA KRAFQPGSNP PPYSQFLSRR NEIPLMRFST PRPRRHTRSA QDHADPDPLR


121
VLKPRLRLTP APASCSQELP SDEDDGAVAS DPLRVVLGRR PHARAAGAGG ERCRPGPQLS


 







Amino acid sequence of Macropus eugenii


(wallaby) FGF23 (SEQ ID NO: 261)


(Ensembl accession no. ENSMEUP00000003725,


which is hereby incorporated by reference in its entirety):








1
ALMIRSEDAG LVIISGVMSR RYLCMDLRGN IFGSHFFSPD NCRFKHRTLE NGYDIYHSPQ


61
NNLLISLGKA KRAFLPGMNP PPYSQFLSRR NEIPIIHFNT PEPRRHTRSA ENSPDLDPMN


121
VLKPRPRVTP CSQELRSAEE NSVVDDDPLE VLRNSNRLKP YPGRMSLERC LQVPKAA


 







Amino acid sequence of Taeniopygia guttata


(zebra finch) FGF23 (SEQ ID NO: 262) (GenBank


accession no. XP_002190520








1
MEWRATLQGI PCSSLLLLLC SLKASLAFPN SSPLLSPSWG NGDRLMHLYT DTERSSFHLQ


61
INADGYIDGA PHQTIYSALM IKSEGAGSVI ITGVKSGRYL CMDMKGNIFG SHYFSQEDCM


121
FNHRTLENGY DVYQSPKHHF LVSLGRVKQV FSPGMNPPPY SQFLSRKNEI PLFRFNTPEP


181
HRHTRSADVD PVDPHQILVP QRKTPVFGSL QQQPADFPHM PREPMRINQN DVVNPDDPHA


241
MMEARRYPSP RFYITR


 







Amino acid sequence of Gallus gallus


(chicken) FGF23 (SEQ ID NO: 263)


(GenBank accession no. XP_425663,


which is hereby incorporated by reference in its entirety):








1
MPHTSPCSCL EYMLLVLCIL KAAVAFPNSS PLLNPSWGNG DQLMHLYTST ERNSFHLQIN


61
ADGHINGVPH QTIYSALMIK SEGAGCVIIT GVKSGRYLCM DMKGDIFGSY YFSQEDCVFN


121
QRTLENGYDV YQSPKHNFLV SLGRTKQVFF PGMNPPPYSQ FLSRRNEIPL FRFNTPEPHR


181
NTRSADVDPL DPHQILVPQR KVSALGSQLQ LQMDFSHVPR EPMRVNQNDV VNPDDPHAMM


241
DARRYASPRF YITR


 







Amino acid sequence of Meleagris gallopavo


(turkey) FGF23 (SEQ ID NO: 264) (GenBank


accession no. XP_003202623,


which is hereby incorporated by reference in its entirety):








1
MPHTSPCSCL EYMLLVLCIL KAAVSFPNSS PLLNPSWGNG DQLMHLYTST ERNSFHLQIN


61
ADGHISGVPY QTIYSALMIK SEGAGSVIIT GVKSGRYLCM DMKGDIFGSH YFSQEDCVFN


121
QRTLENGYDV YQSPKHNFLV SLGRTKQVFF PGMNPPPYSQ FLSRRNEIPL FRFNTPEPHR


181
NTRSADVDPM DPHQILVPQR KVSAIESQLQ LQMDFSHVPR EPMRVNQNDV VNPDDPHAMM


241
DARRYASPRF YITR


 







Amino acid sequence of Anolis carolinensis


(green anole) FGF23 (SEQ ID NO: 265)


(GenBank accession no. XP_003221411,


which is hereby incorporated by reference in its entirety):








1
MVQATLYSFL KYMLLATCSW KAIAAFPNAS PLLSLNWGNS DSLLHLYTST ARNSFHLQIH


61
SNGYVDGSPY QTIYSALMIK SEVAGYVIIN GVKSGRFLCM DMNGNIFGSH FFSYEDCTFK


121
HWVLENGYDV YQSPKYNYLV SLGKAKQPLF PNMNPPPYSQ FLSRRNEIPL VQFNTPKPHR


181
HTRSANADPC GSIISSGNIA KENLQLQPLM YNTKMNSNSE DEDPNSAIIN RRFLSPRTDV


241
RS


 







Amino acid sequence of Latimeria chalumnae


coelacanth) FGF23 (SEQ ID NO: 266) (Ensembl


accession no. ENSLACP00000020506,


which is hereby incorporated by reference in its entirety):








1
LESALLAFSM AIFYSFKAVS SFPNSSPLLN PVWGNTDNLI HLYTASETNS FHLQINSDGH


61
VDGTPHQTAY SALLIKSEEA GSVVILGVKS GRYLCMDIKG NIIGLHHFSK EDCTFKQEGL


121
ENGFDVLRSP KHNILVSLDK TKRSYIPGMN LPPYSQFLSR QNEVALINFI NTPDIHRHSR


181
NVDVDPSDPH GMIIQPDVGV SFRKSSSLFS DLPRDSMRTS HNGMDMVDPA DPHGMLDSRR


241
RPSPRFFAR


 







Amino acid sequence of Xenopus silurana


tropicalis (western clawed frog) FGF23(SEQ


ID NO: 267) (GenBank accession no.


XP_002940351,


which is hereby incorporated by reference in its entirety):








1
MTKQQTRLGL VLTVLASIKV ISAFPNSSPI ISGGWGVPDR LMHLYTASDW NSFHLQINHD


61
GSIDGTPTQT IYSAIMIKSE SAGHVVITGV KTNRYLCMDK SGNIFGYHDF NHDDCVFKHE


121
TLENNFDVYH SPKHNFVISL KEPKHHFRLG MDLPPYSQFL SLENEIPITR FNAPEPEMRI


181
PEGNFADPSD IIKNPRNWDF SQSIHNPFQD VWLPFPSGSL PIIRASLPII HNNVINTDDP


241
EEIVKMKRYR YFKR


 







Amino acid sequence of Felis catus (cat)


FGF23 (SEQ ID NO: 268) (Ensembl


accession no. ENSFCAP00000000128,


which is hereby incorporated by reference in its entirety):








1
MSGTRLGLLV SVLCWVVRAY PNTSPLLGSS WGGLTHLYTA TARNSYHLQI HKDGHVDGTP


61
HQTIYSALMI RSEDAGFVVI TGVMSQRYLC MDFRGNIFGS HLFSPESCRF RQRTLENGYD


121
VYHSPQHRFL VSLGPAKRAF LPGTNRMTPA PASCSQELPS AEDSGVVASD PLGVLRGNRV


181
NAHAGGMGVE RCRPFPKFN


 







Amino acid sequence of Pelodiscus sinensis


(Chinese softshell turtle) FGF23(SEQ ID NO:


269) (Ensembl accession no. ENSPSIP00000012755,


which is hereby incorporated by reference in its entirety):








1
MSQPSQCSCL NFMLFVLCSF KAIAAFPFFS SLLNPSWGET DSLIHLYTAT EKNSFHLQIN


61
PDGYVDGTPH QTIYSALMIK SEDAGYVVIS GVKSGRYLCM DIKGNIFGSH YFSQEDCMFK


121
HRTLENGYDV YQSPKHNFLV SLGRNKQAFF PGMNLPPYSQ FLPRRNEIPL IRFNTPEPHR


181
HTRNADVDPL QILIPRGEAF DTGPQRLQTH FDHLPREPMR INPNDVVSPD DPLAMMDVRR


241
NASPRLYITR


 







Amino acid sequence of Mustela putorius


furo (Ferret) FGF23 (SEQ ID NO: 270) (Ensembl


accession no. ENSMPUP00000009243,


which is hereby incorporated by reference in its entirety):








1
MSVTRLGLLV SVLCWVVRAY PNASPLLGSS WGGLTHLYTA TARNSYHLQI HKDGHVDGTP


61
HQTIYSALMI RSEDAGFVVI TGVMSRRYLC MDFRGNIFGS HLFSPESCRF RQRTLENGYD


121
VYHSPQHRFL VSLGQAKRAF LPGTNPPPYS QFLSRRNEIP LIHFNTPRPR RHTRSAEDME


181
HDPLNVLKPR PRMTPAPASC SQELPSAEDN SVVASDPLGV LRGNRVNVHA GGMGVDRCRP


241
LPKFI


 







Amino acid sequence of Microcebus murinus


(Mouse lemur) FGF23(SEQ ID NO: 271)


(Ensembl accession no. ENSMICP00000004444,


which is hereby incorporated by reference in its entirety):








1
MLGACLRLWV CALCSVCGVS VVRAYPNASP LLASSWGGLI HLYTATARNS YHLQIHKDGH


61
VDGTPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHVFSA ESCRFRQRTL


121
ENGFDVYQSP QHHFLVSLGR AKGAFPAGAK PPPFPQFLPR GNEAPGRKTR GPEEKGAPHP


181
LRGVESGGRK GGAPPLCLER LSRARE


 







Amino acid sequence of Pongo abelii


(Orangutan) FGF23 (SEQ ID NO: 272)


(Ensembl accession no. ENSPPYP00000005881,


which is hereby incorporated by reference in its entirety)


(partial sequence corresponding to human


FGF23 residues 23 to 37 and 72 to 251):








1
M--------- ---------- --RN--ESLP CLVFSIG--- ---------- ----------


61
---------- -ALMIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHYFNP ENCRFQHQTL


121
ENGYDVYHSP QHHFLVSLGR VKRAFLPGM- PPPYSQFLSR RNEIPLIHFN TPVPRRHTRS


181
AEDDTERDPL KVLKPRARMT PAPASCSQEL PSSEDNSPMA SDPLGVVRGG RVNTHAGGTG


241
PEGCRPFPKF I


 







Amino acid sequence of Sorex araneus


(Shrew) FGF23 (SEQ ID NO: 273)


(Ensembl accession no. ENSSARP00000007042,


which is hereby incorporated by reference in its entirety)


(partial sequence corresponding to human FGF23


residues 1 to 18, 28 to 70, 106 to 197, and 201 to 235):








1
MWGLRLGLLV GLLGCVDR-- -------ASP MLASSWGGLT HLYTATARNS YHLQIHKDGL


61
VDGSPQQTVY ---------- ---------- ---------- -----HHFSP ESCRFQQRTL


121
ENGYDVYQSP QHRFLVSLGR PKRAFQPGAN PPPYAQFLAR RNEVPLARFH TPAPRRHTRS


181
AHDNGDADPL NVLAPRA--- AAAASCSHEL PSAEDNSVVA SDPLGVIRSN RFRTH


 







Amino acid sequence of Tetraodon nigroviridis


(Tetraodon) FGF23 (SEQ ID NO: 274)


(Ensembl accession no. ENSTNIP00000014355,


which is hereby incorporated by reference in its entirety):








1
MDVNRRIGVK DALLALLLAL LQGCPLGETA PNASPLVGSN WGNPRRYVHL QTSTDMSNFY


61
LEIRLDGTVR KSTARTSYSV ILLKADTRER IAILGVKSNR YLCMDLEGSP FSSPTCIRDD


121
CLFNHSLLEN NRDVYYSSRT GILFNLEGSR QVFVVGQNVP QTSLFLPRTN TVPLERLLLH


181
RDKRNQVVDP SDPHRVAVGR AEEGSDSRAL QEDDADLEVE TEVEVGDDGR NASRERLQAP


241
SDHDPWGVFS SNPGSPRSSG TVG


 







Amino acid sequence of Oreochromis niloticus


(Tilapia) FGF23 (SEQ ID NO: 275)


(Ensembl accession no. ENSONIP00000000020,


which is hereby incorporated by reference in its entirety):








1
MDVNRRMGMR DTVLALFLAV LQGFPLGDTV PNPSPLAGSN WGNPRRYVHL QTSTDLNNFY


61
LEIRLDGSVR KTTSRSTYSV ILLKSEARDR VAILGVKSSR YLCMDLEGNP FSSPVCLRDD


121
CLFNHKLLEN NRDVYYSSRT GILFNLEGSR QVYSVGQNLP QTSLFLPRKN TVPLERLLLH


181
REKRNRGQTE EGSDSRAVPE ELEEREVEME TEIETEVGDD GRNVSREKLA APSSHDPWNV


241
HFSNPASPRS TGTVG


 







Amino acid sequence of Danio rerio


(Zebrafish) FGF23 (SEQ ID NO: 276) (Ensembl


accession no. ENSDARP00000067387,


which is hereby incorporated by reference in its entirety):








1
MRCALSNLHM LHSSVLALWF TALQGLRPAD AAPNPSPLLG SNWGNPRRYI HLQTTSDLNN


61
YYLEISPSGH VRKTTNRGSY SVILLKTESR DRLAIFGVKS NRFLCMDTGG TLFTSTICNK


121
EDCLFHHKLL ENHRDVYYST KHSILLNLDG DKQAFIAGQN LPQSSLFLSE KNTVPLERLQ


181
HRERRNRQVN PTDPLNALRY AEESDSRAAQ EDDGDMDFEP SEGQNISRET LVSPSDDDPW


241
DLLHDTSPGS PRIAAIVG









In certain embodiments according to the present invention, the C-terminal portion of FGF23 of the chimeric protein of the present invention includes a polypeptide sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 233.
It will be understood that the FGF23 according to the present invention may be from a nucleotide sequence that encodes an FGF23 protein (e.g., those encoding orthologs) from a mammal or even a non-mammalian species. For example, a nucleotide sequence encoding a mammalian or non-mammalian FGF23 protein according to the present invention may include, but is not limited to, those FGF-encoding nucleotide sequences shown in Table 8.







TABLE 8







Human FGF23 gene coding sequence (1-251) (SEQ ID NO: 277)


(GenBank accession no. AF263537,


which is hereby incorporated by reference in its entirety)








147
                            ATGT TGGGGGCCCG CCTCAGGCTC TGGGTCTGTG


181
CCTTGTGCAG CGTCTGCAGC ATGAGCGTCC TCAGAGCCTA TCCCAATGCC TCCCCACTGC


241
TCGGCTCCAG CTGGGGTGGC CTGATCCACC TGTACACAGC CACAGCCAGG AACAGCTACC


301
ACCTGCAGAT CCACAAGAAT GGCCATGTGG ATGGCGCACC CCATCAGACC ATCTACAGTG


361
CCCTGATGAT CAGATCAGAG GATGCTGGCT TTGTGGTGAT TACAGGTGTG ATGAGCAGAA


421
GATACCTCTG CATGGATTTC AGAGGCAACA TTTTTGGATC ACACTATTTC GACCCGGAGA


481
ACTGCAGGTT CCAACACCAG ACGCTGGAAA ACGGGTACGA CGTCTACCAC TCTCCTCAGT


541
ATCACTTCCT GGTCAGTCTG GGCCGGGCGA AGAGAGCCTT CCTGCCAGGC ATGAACCCAC


601
CCCCGTACTC CCAGTTCCTG TCCCGGAGGA ACGAGATCCC CCTAATTCAC TTCAACACCC


661
CCATACCACG GCGGCACACC CGGAGCGCCG AGGACGACTC GGAGCGGGAC CCCCTGAACG


721
TGCTGAAGCC CCGGGCCCGG ATGACCCCGG CCCCGGCCTC CTGTTCACAG GAGCTCCCGA


781
GCGCCGAGGA CAACAGCCCG ATGGCCAGTG ACCCATTAGG GGTGGTCAGG GGCGGTCGAG


841
TGAACACGCA CGCTGGGGGA ACGGGCCCGG AAGGCTGCCG CCCCTTCGCC AAGTTCATCT


901
AG


 







Gorilla FGF23 gene coding sequence (1-251) (SEQ ID NO: 278)


(Ensembl accession no. ENSGGOT00000002983,


which is hereby incorporated by reference in its entirety)








1
                            ATGT TGGGGGCCCG CCTCAGGCTC TGGGTCTGTG


35
CCTTGTGCAG CGTCTGCAGC TTGAGCGTCC TCAGAGCCTA TCCCAATGCC TCCCCACTGC


95
TCGGCTCCAG CTGGGGTGGC CTGATCCACC TGTACACAGC CACAGCCAGG AACAGCTACC


155
ACCTGCAGAT CCACAAGAAT GGCCATGTGG ATGGCGCACC CCATCAGACC ATCTACAGTG


215
CCCTGATGAT CAGATCAGAG GATGCTGGCT TTGTGGTGAT TACAGGTGTG ATGAGCAGAA


275
GATACCTCTG CATGGATTTC AGAGGCAACA TTTTTGGATC ACACTATTTC GACCCGGAGA


335
ACTGCAGGTT CCAACACCAG ACGCTGGAAA ACGGGTACGA CGTCTACCAC TCTCCTCAGT


395
ATCACTTCCT GGTCAGTCTG GGCCGGGCGA AGAGAGCCTT CCTGCCAGGC ATGAACCCAC


455
CCCCGTACTC CCAGTTCCTG TCCCGGAGGA ACGAGATCCC CCTCATTCAC TTCAACACCC


515
CCATACCACG GCGGCACACC CGGAGCGCCG AGGACGACTC GGAGCGGGAC CCCCTGAACG


575
TGCTGAAGCC CCGGGCCCGG ATGACCCCGG CCCCGGCCTC CTGTTCACAG GAGCTCCCGA


635
GCGCCGAGGA CAACAGCCCG ATGGCCAGTG ACCCATTAGG GGTGGTCAGG GGCGGTCGAG


695
TGAACACGTA CGCTGGGGGA ACGGGCCCGG AAGGCTGCCG CCCCTTCCCC AAGTTCATCT


755
AG


 







Northern white-cheeked gibbon FGF23 gene coding sequence


(1-251) (SEQ ID NO: 279) (GenBank accession no.


XM_003273701,


which is hereby incorporated by reference in its entirety)








140
                    A TGTTGGGGGC CCGCCTCAGG CTCTGGGTCT GTGCCTTGTG


181
CAGCGTCTGC AGCATGAGCG TCCTCAGAGC CTATCCCAAT GCCTCCCCAC TGCTCGGCTC


241
CAGCTGGGGT GGCCTGATCC ACCTGTACAC AGCCACAGCC AGGAACAGCT ACCACCTGCA


301
GATCCACAAG AATGGCCATG TGGATGGCGC ACCCCATCAG ACCATCTACA GTGCCCTGAT


361
GATCAGATCA GAGGATGCTG GCTTTGTGGT GATTACAGGT GTGATGAGCA GAAGATACCT


421
CTGCATGGAT TTCAGAGGCA ACATTTTTGG ATCACACTAT TTCAACCCGG AGAACTGCAG


481
GTTCCAACAC CAGACGCTGG AAAACGGGTA CGACGTCTAC CACTCTCCTC AGCATCACTT


541
CCTGGTCAGT CTGGGCCGGG CCAAGAGAGC CTTCCTGCCG GGCATGAACC CACCCCCGTA


601
CTCCCAGTTC CTGTCCCGGA GGAACGAGAT CCCCCTACTT CACTTCAACA CCCCCACACC


661
ACGGCGGCAC ACCCGGAGCG CCGAGGACGA CTCGGAGCGG GACCCCCTGA ACGTGCTGAA


721
ACCCCGGGCC CGGATGACCC CGGCCCCGGC CTCCTGCTCA CAGGAGCTCC TGAGCTCCGA


781
GGACAACAGC CCGATGGCCA GCGACCCATT AGGGGTGGTC AGGGGCGGTC GAGTGAACAC


841
GCACGCTGGG GGAACGGGCC CGGAAGGCTG CCGCCCCTTC CCCAAGTTCA TCTAG


 







Rhesus monkey FGF23 gene coding sequence (1-251) (SEQ


ID NO: 280) (GenBank accession no. NM_001194137,


which is hereby incorporated by reference in its entirety)








69
        AT GTTGGGGGCC CGCCTCAGGC TCTGGGTCTG TGCCTTGTGC AGCGTCTGCA


121
GCATGAGCGT CATCAGAGCC TATCCCAATG CCTCCCCATT GCTCGGCTCC AGCTGGGGTG


181
GCCTGATCCA CCTGTACACA GCCACAGCCA GGAACAGCTA CCACCTGCAG ATCCACAAGA


241
ATGGCCACGT GGATGGCGCA CCCCATCAGA CCATCTACAG TGCCCTGATG ATCAGATCAG


301
AGGATGCTGG CTTTGTGGTG ATTACAGGTG TGATGAGCAG AAGATACCTC TGCATGGATT


361
TCAGAGGCAA CATTTTTGGA TCACACTATT TCAACCCGGA GAACTGCAGG TTCCGACACT


421
GGACGCTGGA GAACGGCTAC GACGTCTACC ACTCTCCTCA GCATCACTTT CTGGTCAGTC


481
TGGGCCGGGC GAAGAGGGCC TTCCTGCCAG GCATGAACCC ACCCCCCTAC TCCCAGTTCC


541
TGTCCCGGAG GAACGAGATC CCCCTCATCC ACTTCAACAC CCCCAGACCA CGGCGGCACA


601
CCCGGAGCGC CGAGGACGAC TCGGAGCGGG ACCCCCTGAA CGTGCTGAAG CCCCGGGCCC


661
GGATGACCCC GGCCCCGGCC TCCTGCTCAC AGGAGCTCCC GAGCGCCGAG GACAACAGCC


721
CGGTGGCCAG CGACCCGTTA GGGGTGGTCA GGGGCGGTCG GGTGAACACG CACGCTGGGG


781
GAACGGGCCC GGAAGCCTGC CGCCCCTTCC CCAAGTTCAT CTAG


 







Crab-eating macaque FGF23 gene coding sequence (1-251) (SEQ


ID NO: 281) (GenBank accession no. ENSMMUT00000020999,


which is hereby incorporated by reference in its entirety)








1
               ATGTTG GGGGCCCGCC TCAGGCTCTG GGTCTGTGCC TTGTGCAGCG


47
TCTGCAGCAT GAGCGTCATC AGAGCCTATC CCAATGCCTC CCCATTGCTC GGCTCCAGCT


107
GGGGTGGCCT GATCCACCTG TACACAGCCA CAGCCAGGAA CAGCTACCAC CTGCAGATCC


167
ACAAGAATGG CCACGTGGAT GGCGCACCCC ATCAGACCAT CTACAGTGCC CTGATGATCA


227
GATCAGAGGA TGCTGGCTTT GTGGTGATTA CAGGTGTGAT GAGCAGAAGA TACCTCTGCA


287
TGGATTTCAG AGGCAACATT TTTGGATCAC ACTATTTCAA CCCGGAGAAC TGCAGGTTCC


347
GACACTGGAC GCTGGAGAAC GGCTACGACG TCTACCACTC TCCTCAGCAT CACTTTCTGG


407
TCAGTCTGGG CCGGGCGAAG AGGGCCTTCC TGCCAGGCAT GAACCCACCC CCCTACTCCC


467
AGTTCCTGTC CCGGAGGAAC GAGATCCCCC TCATCCACTT CAACACCCCC AGACCACGGC


527
GGCACACCCG GAGCGCCGAG GACGACTCGG AGCGGGACCC CCTGAACGTG CTGAAGCCCC


587
GGGCCCGGAT GACCCCGGCC CCGGCCTCCT GCTCACAGGA GCTCCCGAGC GCCGAGGACA


647
ACAGCCCGGT GGCCAGCGAC CCGTTAGGGG TGGTCAGGGG CGGTCGGGTG AACACGCACG


707
CTGGGGGAAC GGGCCCGGAA GCCTGCCGCC CCTTCCCCAA GTTCATCTAG


 







Chimpanzee FGF23 gene coding sequence (1-251) (SEQ ID NO:


282) (GenBank accession no. XM_001157070,


which is hereby incorporated by reference in its entirety)








141
                      ATGTTGGGGG CCCGCCTCAG GCTCTGGGTC TGTGCCTTGT


181
GCAGTGTCTG CAGCGTGAGC GTCCTCAGAG CCTACCCCAA TGCCTCCCCA CTGCTCGGCT


241
CCAGCTGGGG TGGCCTGATC CACCTGTACA CAGCCACAGC CAGGAACAGC TACCACCTGC


301
AGATCCACAA GAATGGCCAT GTGGATGGCG CACCCCATCA GACCATCTAC AGTGCCCTGA


361
TGATCAGATC AGAGGATGCT GGCTTTGTGG TGATTACAGG TGTGATGAGC AGAAGATACC


421
TCTGCATGGA TTTCAGAGGC AACATTTTTG GATCACACTA TTTCAACCCG GAGAACTGCA


481
GGTTCCAACA CCAGACGCTG GAAAACGGGT ACGACGTCTA CTACTCTCCT CAGTATCACT


541
TCCTGGTCAG TCTGGGCCGG GCGAAGAGAG CCTTCCTGCC AAGCATGAAC CCACCCCCGT


601
ACTCCCAGTT CCTGTCCCGG AGGAACGAGA TCCCCCTAAT TCACTTCAAC ACCCCCATAC


661
CACGGCGGCA CACCCGGAGC GCCGAGGACG ACTCGGAGCG GGACCCCCTG AACGTGCTGA


721
AGCCCCGGGC CCGGATGACC CCGGCCCCGG CCTCCTGTTC ACAGGAGCTC CCGAGCGCCG


781
AGGACAACAG CCCGATGGCC AGTGACCCAT TAGGGGTGGT CAGGGGCGGT CGAGTGAACA


841
CGCACGCTGG GGGAACGGGC CCGGAAGGCT GCCGCCCCTT CCCCAAGTTC ATCTAG


 







White-tufted-ear marmoset FGF23 gene coding sequence


(1-251) (SEQ ID NO: 283) (GenBank


accession no. XM_002752235,


which is hereby incorporated by reference in its entirety)








1
ATGTTGGGGG CCCGCCTCAG GCTCTGGGTC TGTGCCTTGT GCAGCGTCTG CAGCATGAGC


61
GTCCTCAGAG CCTATCCCAA TGCCTCCCCA CTGCTTGCCT CCAGCTGGGG TGGCCTGATC


121
CACCTGTACA CAGCCACAGC CAGGAACAGC TACCACCTGC AGATCCACAA GAATGGCCAT


181
GTGGATGGCG CACCCCATCA GACCATCTAC AGTGCCCTGC TGATCAGATC AGAGGATGCT


241
GGCTTTGTGG TGATTACAGG TGTGATGAGC AGAAGATACC TCTGCATGGA TTTCAGAGGC


301
AACATTTTTG GATCACACTA TTTCAACCCG GAGAACTGCA GGTTCCGACC CCAGAGGCTG


361
GAGAACGGGT ACGACGTCTA CCAGTCTCCT CAGCATCACT TCCTGGTCAG TCTGGGCCGG


421
GCGAAGAGGG CCTTCCTGCC AGGCATGAAC CCACCCCCGT ACTCCCAGTT CCTGTCCCGG


481
AGGAACGAGA TCCCCCTCAT TCACTTCAAC ACCCCCAAAC CGCGGCGGCA CACCCGGAGC


541
GCCGAGGACG ACCCGGAGCT AGACCCCCTG AACGTGCTGA AGTCCCGGGT CCGGATGACC


601
CCGGCCCCGG CCTCCTGCTC GCAGGAGCTC CTGAGCGCCG AGGACAACAG CCCGGTGGGC


661
AGCGACCCCT TAGGGATGGT CCGGGGTGGT CGGGTGAACA GCCACGCTGA GGGAACAGGC


721
CCAGAAGGCT GCAGCCCCTT CCCCAAGCTC ATCTAG


 







Elephant FGF23 gene coding sequence (1-251) (SEQ ID NO:


284) (GenBank accession no. XM_003410629,


which is hereby incorporated by reference in its entirety)








1
ATGTTGGGGG CCCGCCTCAG GCTCTGGGTC TGCACCCTGT GCAGTGCCTG CAGCATGTGC


61
AGTGTCAGAG CCTATCCCAA TGCCTCCCCG CTGCTCCACT CCAGCTGGGG TGGCCTGACC


121
CACCTGTACA CAGCCACCGC CAGGAACAGC TACCACCTGC AGATCCACAA GGACGGCCAT


181
GTGGATGGTA CGCCGGACCA GACCATCTAC AGTGCCCTGA TAATCAGATC AGAGGAGGCC


241
GGCTTCGTGG TGATTACAGG GGTGATGAGT AGGAGATACC TCTGTATGGA TTTCAGAGGC


301
AACATTTTTG GATCGCATTA CTTCAACCCA GAGAACTGCA GGTTCAAACA CTGGACGCTG


361
GAAAATGGAT ATGACGTCTA TCACTCTCCT CAGCATCATT TCCTGGTCAG TCTGGGTCGC


421
GTGAAGAAGG CCTTCCTGCC AGGCATGAAC CCACCACCTT ACTCTCAGTT CCTGTCCCGG


481
AGGAATGAGA TCCCCTTGAT TTACTTCAAC ACCCCCAAGC CCCGGCGGCA CACCCGGAGT


541
GCCGAGGATG ACTCTGAACG GGACCCACTG AATGTGCTGA AGCCCCGGCC CCGTATGACA


601
CCTGCTCCAG CTTCTTGCTC CCAGGAACTC CTGAGTGCTG AAGACAACAG CGTGGTGGCC


661
AATGACCCTT TAGGAGTGGT CAGAAGCAAT AGGGTCAACA CACATGCTGG TGGGATAGGT


721
GTGGAAAGGT GCCGCCCCTT CCCCAAGTTC ATCTAG


 







Lesser hedgehog tenrec FGF23 gene coding sequence (1-250)


(SEQ ID NO: 285) (Ensembl accession no. ENSETET00000001609,


which is hereby incorporated by reference in its entirety)








1
ATGTTGGGGG CCCACCTCAG ACTCTGGGTC TGTGCCTTGT GCAGTGTGAG CGCCATGTAC


61
CACGTCAGAG CCTACCCCAA CGCCTCCCCG CTCCTGGGTA CCAGCTGGGC TGGCCTGACC


121
CACCTGTACA CGGCGACAGC CAGGAACAGC TTCCACCTGC AGATCCACAA GGATGGCCAC


181
GTGGACGGCA CCCCCCACCA GACCATCTAC AGTGCCCTGA TGATCCGATC AGAGGACTCT


241
GGCTTCGTGG TGATCACAGG GGTGATGAGC AGGAGATACC TGTGTATGGA TTTCAGAGGC


301
AACATTTTTG GATCGCACTA CTTCACTGCG GACAGCTGCA GGTTCAGACA GCGGACGCTG


361
GAGAACGGCT ATGACGTCTA CCACTCTCCT CAGCATCATT TCCTGATCAG CCTGGGCCGG


421
GCCAAGAGGG TCTTCCTGCC CGGCATGAAC CCGCCGCCTT ACTCCCAGTT CCTGTCCCGA


481
AGGAATGAGA TCCCCCTGAT TCACTTCAAC ACCCCCAGGC CCCGGCGGCA CACACGGAGT


541
GCCGAGGAGG AAGTGGAGCA GGATCCGCTG AACGTGCTGA AGCCCAGGCC CCGGATGACG


601
CCGGCTCCAG CCTCCTGCTC CCAGGAGCTG CCCAGTGCCG AAGACAACAG CGCCCTGGCC


661
AGCGACCCGC TGGGAGTGGT CAGAGGCAAA AAGCTCAACA CCCATGCTGT GGGCATGGGC


721
GCGGAAAGAT GCCGCCCCTT TCCCAAGTTC


 







Hedgehog FGF23 gene coding sequence (1-63 and 73-244)


(SEQ ID NO: 286) (Ensembl accession


no. ENSEEUT00000007917,


which is hereby incorporated by reference in its entirety)








1
ATGTTGGGGG CCCACCTGGG TCTGGTGGTC TGCGCCCTGG TCAGCAGAGC CTATCCCAAT


61
GCCTCGCCAC TGCTGGGCTT CAGCTGGGGG GGCCTGACAC ATCTGTACAC GGCCACAGCC


121
AGGAACAGCT ACCACCTGCA GATCCACAAG GACGGCCACG TGGACGGCTC GCCTCAGCAG


181
ACCATCTACA ---------- ---------- -----TGCTG GTTTCGTGAT GATCACAGGC


241
GTGATGAGTA GGCGCTACCT CTGCATGGAC TTCAGGAGCA ACATCTTTGG ATCGCATCAC


301
TTCGCCCCTG AGAGCTGCAG GTTCAGACAT CGGACACTGG AAAACGGCTA TGACGTCTAC


361
CACTCCCCCC AGCACCATTT CCTGGTCAGC CTGGGCCGGG CCAAGCGGGC CTTCCTGCCG


421
GGCACCAACC CCCCACCATA CTCCCAGTTT TTGTCCCGGA GGAACGAGGT TCCCCTCATC


481
CACTTCAACA CCCCCAGGCC CAGGCGTCAC ACCCGCAGCG CCGAGGACAA CTCAGAGCTG


541
GATCCCCTGA ACGTGCTGAA GCCCAGGCCC CGCATGACCC CCGCCCCAGC CTCCTGCTCC


601
CAGGAGCTTC CGAGCGCTGA GGACAACAGC ATGGTGGCCA GTGACCCACT GGGTGTGGTC


661
AGAGCCAACA GAGTGAACAC ACACGCAGGG GGCCTGGGTG TGGACAAGTG CCGCCCCTTC


721
CCCAAGTTTA TCTAG


 







Bushbaby FGF23 gene coding sequence (1-252) (SEQ ID NO:


287) (Ensembl accession no. ENSOGAT00000005213,


which is hereby incorporated by reference in its entirety)








1
ATGCTGGGGA CCTGCCTCAG GCTCTGGGTC TGTGCCCTGT GCAGTGTTTG CAGCGTGAGC


61
ATTGTCAGAG CCTATCCCAA CGCCTCCCCA CTGCTCAGCT CCAGCTGGGG TGGCCTGACC


121
CACCTGTACA CGGCCTCGGC CAGAAACAGC TACCACCTGC AGATCCACAA GGATGGCCAT


181
GTGGACGGCA CACCCCACCA GACCATCTAC AGCGCCCTAA TGATCAGGTC AGAGGATGCT


241
GGCTTCGTGG TGATTACAGG CGTGATGAGC AGAAGATACC TCTGTATGGA TTTCAAAGGC


301
AACATTTTTG GATCACACTC CTTCCACCCC GAGAGCTGCA GGTTCAGACA CCGGACTCTG


361
GAGAACGGCT ATGACGTCTA CCTCTCGCCG CAGCATCACT TCTTGGTCAG CCTGGGCCGC


421
TCCAAGAGGC CCTTCCTGCC GGGCATGAAC CCGCCCCCCT TCTCCCAGTT CCTGTCGCGG


481
AGGAACGACA TCCCGCTCAT TCACTTCAAC ACCCCCCGCC CGCGGAGACA CACCCGCAGC


541
GCCGAGGACA ACGACTCGGA GCTCGACCCC CTGAACGTGC TGAAGCCGCG GCCCCGGGCC


601
ACCCCGGGCC CCGCCTCCTG CTCGCAGGAG CTCCCCAGCG CCGAGGACAA CAGCCTGGTG


661
GCCAGCGACC CTTTAGGGGT GGTCCGGGGC AACAGGGTGA ACGCTCACGC CGGGAGGGCC


721
GGCCTGGACA GGTGCCGCCC CTTCCCCAGG TATTTCTAG


 







Rabbit FGF23 gene coding sequence (1-252) (SEQ ID NO: 288)


(GenBank accession no. XM_002712826,


which is hereby incorporated by reference in its entirety)








1
ATGTTAGGGG CCCGGCTCCT CCGGCTCTTG GTCTGTGCCC TGGGCAGTGT GTGCAGCTGG


61
TGTGTGGTCC GAGCCTACCC TGACACCTCC CCGCTGCTCA GCTCCAGCTG GGCTGGCCTG


121
ACCCACCTGT ACACGGCCAC CGCCAGAAAC AGCTACCACC TGCAGATCCA CAAGGACGGC


181
CAAGTGGATG GCACACCTCA TCAGACCATC TACAGTGCCC TGATGATCAG ATCGGAGGAT


241
GCTGGCTTCG TGGTGATAAC AGGTGTCATG AGCAGGAGGT ACCTCTGTAT GGATTTCAGA


301
GGCAACATTT TTGGATCGCA TTACTTCGAC CCCCAGAACT GCAGGTTCAG ACACAGGACG


361
CTGGAAAACG GGTACGACGT CTACCACTCT CCGGAGCATC ACTTCCTGGT CAGCCTGGGC


421
CGGGCCAAGA GGCCCTTCCT GCCAGGCATG AACCCGCCAC CCTATTCCCA GTTCCTGTCC


481
CGGAGGAACG AGATCCCCCT GATCCACTTC AACACGCCGA GGCCGCGAAG GCACACCCGG


541
AGCGCCGAGG ACGCCTGGGA GCAGGACCCG CTGAACGTGC TGAAGCCCAG GTTCCGGCTG


601
ACCCCGGCCC CAGCCTCCTG CTCACAGGAG GCCCCAAGTG CTGAAGACAA TGGCCTGGTG


661
GCCAGCGACC CCTTCGGAGT GCTCCGGGGC AATAGGGTGA ACATGCACGG GGACAGGATG


721
GGCCCGGAAA GGTGCCACCA TTTCCCCAAG TTCATCTAG


 







Horse FGF23 gene coding sequence (1-246) (SEQ ID NO: 289)


(GenBank accession no. XM_001491419,


which is hereby incorporated by reference in its entirety)








1
ATGTCAGGGC CCTGCCTTGG GCTCCTGGTC TACGTCCTGT GCTCCGCAGT GAAAGCCTAT


61
CCCAACGCCT CCCCGCTGCT AGACTCCAGC TGGGGCAGCC TGACCCACCT GTACACGGCC


121
ACAGCCAGGA ACAGCTACCA CCTGCAGATC CACAAGGATG GCCACGTGGA TGGCACACCC


181
CATCAGACCA TCTACAGTGC CCTGATGATC AGATCAGAGG ATGCTGGCTT TGTGGTGATA


241
ACAGGTGTGA TGAGCAGGAG ATACCTCTGC ATGGACTTCA GAGGAAACAT TTTTGGATCA


301
CATCACTTCA GCCCCGAGAG CTGCAGCTTC CGACAGCGGA CGCTGGAGAA CGGCTACGAC


361
GTGTACCACT CGCCGCAGCA TCGCTTCCTC GTCAGCCTGG GCCGCGCCAA GAGGGCCTTC


421
CTGCCCGGCA CGAACCCCCC GCCCTACTCG CAGTTCCTGT CCCGGAGGAA CGAGATCCCC


481
CTGGTCCACT TCAACACCCC GCGGCCGCGG CGGCACACGC GCAGCGCCGA GGACAACTCG


541
GAGCGCGACC CGCTGAACGT GCTGAAGCCC CGGCCCCGCA TGACCCCCGC GCCGGCCTCC


601
TGCTCCCAGG AGCTCCCGAG CGCCGAGGAC AACAGCGTGC TGGCCAGCGA CCCCTTAGGG


661
GTGGTCCGTG GCAACAGGGT GAACACGCAC GCGGGGGGCG CGGGCGTGGA GCGCTGCCGC


721
CCCTTCCCCA AGTTCTTCTA G


 







Giant panda FGF23 gene coding sequence (1-245) (SEQ ID NO:


290) (GenBank accession no. XM_002920450,


which is hereby incorporated by reference in its entirety)








1
ATGTCAGGGA CCCGCCTTGG GCTGCTGGTC TCTGTCCTGT GCTGGGTAGG CAGAGCCTAT


61
CCCAACACCT CCCCACTGCT CGGCTCCAGC TGGGGTGGCC TGACCCACCT GTACACAGCC


121
AGCGCCAGGA ACAGCTACCA CCTGCAGATC CACAAGGACG GCCATGTGGA TGGCACACCC


181
CATCAGACCA TCTACAGTGC CCTGATGATC AGGTCAGAGG ATGCCGGCTT TGTGGTGATA


241
ACAGGTGTGA TGAGTAGGCG ATACCTCTGT ATGGACCTCA GAGGCAACAT CTTTGGATCC


301
CACCTCTTCA GCCCGGAGAG CTGCAGGTTC CGACAGCGGA CGCTGGAAAA CGGCTACGAC


361
GTGTACCACT CGCCGCAGCA CCGCTTCCTC GTCAGCCTGG GCCAGGCCAA GAGGACCTTC


421
CTGCCGGGGA CCAACCCGCC GCCCTACTCC CAGTTCCTGT CCCGGAGGAA CGAGATCCCC


481
CTCATCCACT TCAACACCCC CAGGCCAAGG CGGCACACGC GCAGCGCCGA GGACACGGAG


541
CGCGACCCGT TGAACGTGCT GAAGCCCAGG CCCCGCATGA CCCCCGCCCC GGCCTCCTGC


601
TCCCAGGAGC TCCCGAGCGC CGAGGACAAC AGTGTGGTGG CCAGCGACCC GTTAGGGGTG


661
CTCAGAGGCA ACCGGGTGAA CGCGCACGCC GGGGGGATGG GCGTGGACAG GTGCCGCCCC


721
TTCCCCAAGT TCATCTAG


 







Pika FGF23 gene coding sequence (1-250) (SEQ ID NO: 291)


(Ensembl accession no. ENSOPRT00000007149,


which is hereby incorporated by reference in its entirety)








1
ATGCTGGGGG GGCTGGGGCT GTGGGTCTGT GTCCTGGGCA GTGTGTGCAG CTGGCGTGGG


61
GTCCGTGCCT ATCCCGACAC CTCCCCGCTG CTCGGCTCCA GCTGGACTGG CCTGACCCAC


121
CTGTACACGG CCACCGCCAG GAACAGCTTC CACCTGCAGA TCCACAAGGA TGGCCATGTG


181
GATGGCACAC CCCAGCAGAC CATCTATAGT GCCCTGATGA TCAGATCAGA GGATGCCGGC


241
TTCGTGGTGA TAACAGGTGT CATGAGCAGG AGGTACCTCT GTATGGATTT CAGAGGCAAC


301
ATCTTCGGAT CGCATTACTT CGAGCCACAG AACTGCAGGT TCCAGCAGAG GACGCTGGAG


361
AACGGCTACG ACATCTACCA CTCTCCGCAG CACGACTTCC TGGTCAGCCT AGGTCGGGCC


421
AAGAGGCCGT TCCTGCCAGG CATGAACCCG CCACCCTACT CCCAGTTCCT GTCTCGGAGG


481
AACGAGATTC CGCTGATCCT CTTCAACACG CCCAGGCCTC GGAGGCACAC CCGCAGCGCG


541
GAGGAGGGCT GGGAGCGGGA CCCTCTGAAT GTGCTGAAGT CCAGGCCCCG AATGACCCCG


601
GCCCCAGCCT CCTGCTCGCG GGAGGCCCCC AGTGCCGAAG ACGACGGCCT GCTGGCCAGT


661
GACCCCATGG GAGTGCTCAG AGGCCATAGG GTGGATGTGC ACGGGGGTGG GACGGGTAGG


721
GACAGGTGCC GCCCGTTCCC CAGGTTCATC TAG


 







Cattle FGF23 gene coding sequence (1-245) (SEQ ID NO:


292) (GenBank accession no. XM_002687880,


which is hereby incorporated by reference in its entirety)








1
ATGCTGGGGG CCCGCCTGGG GCTCTGGGTC TGCACCCTGA GCTGTGTGGT CCAAGCCTAT


61
CCCAACAGCT CCCCGCTGCT GGGCTCCAGC TGGGGCGGCC TGACCCACCT GTACACGGCC


121
ACGGCCAGGA ACAGCTACCA CCTGCAGATC CACGGAGACG GGCACGTAGA TGGCTCCCCG


181
CAGCAGACTG TCTACAGCGC CCTGATGATC AGGTCGGAGG ATGCCGGCTT CGTGGTGATA


241
ACAGGTGTGA TGAGCAGGCG GTACCTCTGC ATGGACTTCA CAGGCAACAT TTTTGGATCC


301
CATCACTTCA GTCCGGAGAG CTGCCGGTTC CGGCAGCGGA CACTGGAGAA CGGCTACGAC


361
GTGTACCACT CGCCGCAGCA CCGCTTCCTC GTCAGCCTGG GCCGGGCCAA GCGCGCCTTC


421
CTGCCGGGCA CCAACCCGCC CCCATACGCG CAGTTCCTGT CGCGCAGGAA CGAGATCCCG


481
CTGCCGCACT TCGCCGCCAC CGCGCGGCCC CGGCGCCACA CGCGCAGCGC ACACGACAGC


541
GGGGACCCGC TCAGCGTGCT CAAGCCGCGC GCCCGCGCCA CGCCCGTGCC CGCCGCCTGC


601
TCCCAGGAGC TGCCCAGCGC CGAGGACTCC GGCCCTGCCG CCAGCGACCC GCTCGGGGTG


661
CTCCGCGGAC ACCGCCTGGA CGTGCGCGCC GGCTCCGCGG GCGCCGAGCG CTGCCGGCCC


721
TTCCCCGGCT TCGCCTAG


 







Pig FGF23 gene coding sequence (1-244) (SEQ ID NO: 293)


(GenBank accession no. XM_001926525,


which is hereby incorporated by reference in its entirety)








1
ATGCTGGGGG CCCGCCTCGG GCTCTGGGTC TGCACCCTGT GCTGTGCGGC CAGAGCCTAT


61
CCCGACACCT CCCCGCTGCT GAGCTCTGGC TGGGGCGGCC TGACCCACCT GTACACGGCC


121
ACGGCCAGGA ACAGCTACCA CCTGCAGATC CACAAGGATG GCCACGTGGA TGGCTCACCC


181
CAACAGACCA TCTACAGTGC CCTAATGATC AGGTCGGAGG ACGCAGGCTT CGTGGTCATA


241
ACAGGCGTGA TGAGCAGGAG ATACCTCTGC ATGGACTTAA GGGGCAACAT TTTTGGATCG


301
CTGCACTTCA GCCCCGAGAG CTGCAGGTTC CGGCAGCGGA CGCTGGAGAA CGGCTACGAC


361
GTGTACCACT CGCCGCACTA CCGCTTCCTC GTCAGCCTGG GCCGGGCCAA GCGGGCCTTC


421
CTGCCGGGTA CCAACCCGCC CCCGTACGCG CAGTTCTTGT CGCGCAGGAA CGAGATCCCG


481
CTGCTGCACT TCGCCACCGC GCGGCCCCGG CGCCACACGC GCAGCGCGCA CGACGGCGGG


541
GACCCGCTGA GCGTCCTGAA GCCGCGCGCG CGCGCCACGC CCGCGCCCGT CTCCTGCTCC


601
CGCGAGCTGC CCAGCGCCGA GGACGGCGGC CCCGCGGCCA GCGACCCGCT CGGGGTGCTC


661
CGGGGCCAGC GGCTGGACGC GCGCGCTGGG GTGGGGGGCG CCGAGCGCTG CCGGCCCTTC


721
CCCAGCTTCG CCTAG


 







Dog FGF23 gene coding sequence (1-312) (SEQ ID NO: 294)


(GenBank accession no. XM_849487,


which is hereby incorporated by reference in its entirety)








1
ATGTGGACAG TGGAGTTTTT CCTGTTTGAT GTCACAGGGC CACCCTTTAA AAGTCTGAGG


61
GAAAAAAGGA GGGAATCTAG CCTGGGACTT TCACGCAAGA TACCCACAAA GAAGAGGAGA


121
AAAAGGCCTG TGAGGCACAG CCGGGGAATC AAGGAGGCAG TGTCAGGTTT CAAACTCCAG


181
CCAGCCATTC AGAGAGCTGT GATGTCTGGC ACCCGCCTTG GATTCCTGGT CTCTGTCCTG


241
TGCTGGGTAG TCAGAGCCTA TTCCAACACC TCCCCGCTGC TCGGCTCCAG CTGGGGTAGC


301
CTAACCCACC TGTATACGGC CACAGCCAGG AACAGCTACC ACCTGCAGAT CCACAAGGAC


361
GGCCATGTGG ATGGCACACC TCATCAGACC ATCTACAGTG CCTTGATGAT CCGGTCAGAG


421
GATGCCGGCT TTGTGGTGAT AACAGGTGTG ATGAGTAGGA GGTACCTCTG TATGGACTTC


481
AGAGGCAACA TCTTTGGATC ACACCTCTTC AGCCCGGAGA GCTGCCGGTT CCGACAGCGG


541
ACGCTGGAGA ACGGCTACGA CGTGTACCAC TCCCCGCAGC ACCGCTTCCT CGTCAGCCTG


601
GGCCAGGCCA AGAGGGCCTT CCTGCCCGGC ACCAACCCGC CGCCCTACTC GCAGTTCCTG


661
TCCCGGAGGA ACGAGATCCC CCTCGTGCAC TTCCACACGC CCAGGCCGCG GCGGCACACG


721
CGCAGCGCCG AGGCCCCGGA GCGCGACCCG CTGAACGTGC TGAAGCCCAG GCCGCGCTTG


781
GCCCCCGCCC CGGCCTCCTG CTCGCAGGAG CTCCCGAGCG CCGAGGACCC CGGCGCGCCG


841
GCCAGCGACC CGCTCGGGGT GCTCAGGGGC CACAGGGCCA ACGCGCGCGC CGGCGGGGTG


901
GGCGTGGACA GGTGCCGCGC CTTCCCCACG CCCATCTAG


 







Domestic guinea pig FGF23 gene coding sequence (1-243)


(SEQ ID NO: 295) (GenBank accession no.


XM_003463298,


which is hereby incorporated by reference in its entirety)








1
ATGCTGGGGA CCTGCCTTGG GCTCCTGGCC TGCACCGTGA GCTTAGTAGG AGCCTATCCT


61
GATGCCTCCC CATTGCTCAC CTCCAGCTGG GGTGGCCTGA TCCATCTGTA CACGGCCACA


121
GCCAGAAACA GCTACCATCT GCAGATCCAC AAAGATGGCC ACATAGATGG TGCACCCTAT


181
CCGACCATCT ACAGTGCCCT GATGATCAGA TCAGAAGATG CTGGGTTCGT CGTGATAACA


241
GGGGTCACAA GCAGGAGATT CCTCTGCATG GATTTCAGAG GCAACATTTT TGGATCTCAC


301
CACTTCAATC CCCAAGACTG CCGATTCCAA CACCGCACGC TGGAAAACGG TTACGACGTC


361
TACCTCTCTC CCGAGCACCA CTTTCTGATC AGCCTGGGCA GGACCAAGAA GTTCTTCCTG


421
CCGGGCACCA ACCCACCGCC CTACTCCCAG TTCCTGTCGC GCAGGAACGA GCTGCCCCTG


481
GCCCGCTTCG TCACGCCCGG GCCGCGGCGA CACACGCGCA GCGCGGAGGA GGACCAGGGC


541
CGCGACCCGC TGAGCGTGCT CAAGCTTCGG CCCCGCGCCA CGCCCGCGCC CGCCTCGTGC


601
TCGCAGGAGC TGCCCAGCGC GGAGGACGCG GCCCAGGCCA GCGACCCCCT GGGCGTGCTG


661
CGGGGCGCCA GGGTGCACGC GCACGGCGGG CCGCGCCCCG CGAGGTGCCG CCCGGGACCC


721
GGGGCCAAGT AA


 







Chinese hamster FGF23 gene coding sequence (1-251) (SEQ


ID NO: 296) (GenBank accession no. XM_003496084,


which is hereby incorporated by reference in its entirety)








1
ATGCTGGGGA CCTGCCTCAG ACTCCTGGTG GGTGTTCTGT GTAGTGCCTG CAGCCTGGGC


61
ACTGTTAGAG CCTATCCTGA CACCTCCCCA CTGCTCGGCT CCAATTGGGG CAGCCTGACC


121
CACCTGTACA CAGCTACAGC CAGGAACAGT TATCACCTAC AGATCCACAA GGATGGCCGT


181
GTAGATGGCA CACCCCATCA GACCATCTAC AGTGCCCTGA TGATTAGATC AGAGGATGCT


241
GGCTTCGTGA TCATAACAGG AGCTGTGACT AGAAGGTTCC TTTGTATGGA TCTCAGGGGC


301
AACATTTTTG GATCGCATCA CTTCAGCCCG GAGAACTGCA GGTTCCGCCA GCGGACTCTG


361
GAGAATGGCT ATGACGTCTA CCTGTCGCCA CAGCATCACT ACCTGGTGAG CCTGGGCCGC


421
GCCAAGCGCC CCTTCGAGCC CGGCACCAAC CCGCCTCCCT TCTCGCAGTT CCTGGCGCGC


481
AGGAACGAGG TCCCGCTGCT GCGCTTCCAT ACCGCACGGC CACGGCGCCA CACGCGCAGC


541
GCCGAGGACC CTCCCGAGTG GGACCCACTG AACGTGCTCA AGCCGCGGCC CCGTGCCACG


601
CCCGTGCCCG TGTCCTGCTC GCGGGAGCTG CCGAGCGCCG AGGAAGGTGA CCTCGCGGCC


661
AGTGACCCAC TGGGCGTCCT GCGCAGAGGC CGCGGGGATG CTCGCGGGGG CGCAGGAGGC


721
GTGGACCGGT GCCGTCCCTT TCCCAGATTC GCCTAG


 







Tree shrew FGF23 gene coding sequence (1-180) (SEQ ID


NO: 297) (Ensembl accession no. ENSTBET00000016365,


which is hereby incorporated by reference in its entirety)








1
GCCCTGCTGA TCAGGCCGGA GGAGGCTGGC TTCGCGGTGA TCACGGGCGT GATGAGCAGG


61
AGATACCTCT GCATGGATTT CAGGGGCAAC ATTTTCGGAT CACACCTCTT CAGCCCGGAG


121
AGCTGCAGGT TCCGGCAGCG CGCCCTGGAG AACGGCTACG ACGTCTACCA CCACCCGCAG


181
CACCACTTCC TGGTCAGCCT GGGCCGGCCC AAGAGGGCCT TCGTGCCAGG CACGAACCCG


241
CCCCCCTACT CCCAGTTCCT GGCCCGGAAG AACGAGATCC CGCTCATCCA CTTCAACACC


301
CCGAAGCCGC GGCGGCACAC CCGCAGCGCA GAGGACAACT CGGGGCGCGA CCCGCTGAAC


361
GTGCTGAAGC CCCGGCCGCG CATGACCCCG GCGCCCGCCT CCTGCTCGCA GGAGCTCCCG


421
AGTGCCGAGG ACAACAGCGT GGTGGCCAGC GACCCCCTGG GAGTGCTCAG GGGCAACAGG


481
GTGAACACGC ACGCGGGGGG CTGGGGCGTG GACCGCTGCC GCCCCTTCCC CAGGTTTATC


541
TAG


 







Norway rat FGF23 gene coding sequence (1-251) (SEQ


ID NO: 298) (GenBank accession no. NM_130754,


which is hereby incorporated by reference in its entirety)








1
ATGCTGGGGG CCTGCCTCAG ACTCCTGGTG GGCGCTCTGT GCACCGTCTG CAGCTTGGGC


61
ACTGCTAGAG CCTATTCAGA CACTTCCCCA CTGCTTGGCT CCAACTGGGG GAGCCTGACC


121
CACCTGTACA CAGCTACAGC CAGGAACAGC TATCACCTAC AGATCCATAG GGATGGCCAT


181
GTAGACGGAA CACCCCATCA GACTATCTAC AGTGCCCTGA TGATCACATC AGAGGATGCT


241
GGCTCCGTAG TGATAATAGG GGCCATGACC AGAAGGTTCC TTTGTATGGA TCTCCGCGGC


301
AACATTTTTG GATCGTATCA CTTCAGCCCG GAGAACTGCA GATTCCGCCA GTGGACGCTA


361
GAGAACGGCT ACGACGTCTA CCTGTCACCG AAGCATCACT ACCTGGTGAG CTTGGGCCGC


421
TCCAAGCGCA TCTTCCAGCC CGGTACCAAC CCGCCGCCCT TCTCGCAGTT CCTGGCGCGC


481
AGGAACGAGG TCCCGCTGCT GCACTTCTAC ACCGCGCGCC CACGGCGCCA CACGCGCAGC


541
GCCGAGGACC CGCCCGAGCG CGACCCGCTG AATGTGCTCA AGCCGCGGCC CCGCGCTACT


601
CCCATACCGG TATCCTGCTC GCGAGAGCTA CCGAGTGCAG AGGAAGGTGG CCCCGCGGCC


661
AGCGACCCCC TGGGAGTGCT GCGCAGAGGC CGCGGGGATG CTCGCCGGGG CGCGGGAGGC


721
ACGGATCGGT GTCGCCCCTT TCCCAGGTTC GTCTAG


 







House mouse FGF23 gene coding sequence (1-251) (SEQ ID


NO: 299) (GenBank accession no. BC120605,


which is hereby incorporated by reference in its entirety)








24
                         ATGCTAG GGACCTGCCT TAGACTCCTG GTGGGCGCGC


61
TCTGCACTGT CTGCAGCTTG GGCACTGCTA GAGCCTATCC AGACACTTCC CCATTGCTTG


121
GCTCCAACTG GGGAAGCCTG ACCCACCTGT ACACGGCTAC AGCCAGGACC AGCTATCACC


181
TACAGATCCA TAGGGATGGT CATGTAGATG GCACCCCCCA TCAGACCATC TACAGTGCCC


241
TGATGATTAC ATCAGAGGAC GCCGGCTCTG TGGTGATAAC AGGAGCCATG ACTCGAAGGT


301
TCCTTTGTAT GGATCTCCAC GGCAACATTT TTGGATCGCT TCACTTCAGC CCAGAGAATT


361
GCAAGTTCCG CCAGTGGACG CTGGAGAATG GCTATGACGT CTACTTGTCG CAGAAGCATC


421
ACTACCTGGT GAGCCTGGGC CGCGCCAAGC GCATCTTCCA GCCGGGCACC AACCCGCCGC


481
CCTTCTCCCA GTTCCTGGCG CGCAGGAACG AGGTCCCGCT GCTGCACTTC TACACTGTTC


541
GCCCACGGCG CCACACGCGC AGCGCCGAGG ACCCACCCGA GCGCGACCCA CTGAACGTGC


601
TCAAGCCGCG GCCCCGCGCC ACGCCTGTGC CTGTATCCTG CTCTCGCGAG CTGCCGAGCG


661
CAGAGGAAGG TGGCCCCGCA GCCAGCGATC CTCTGGGGGT GCTGCGCAGA GGCCGTGGAG


721
ATGCTCGCGG GGGCGCGGGA GGCGCGGATA GGTGTCGCCC CTTTCCCAGG TTCGTCTAG


 







Megabat FGF23 gene coding sequence (1-248) (SEQ ID


NO: 300) (Ensembl accession no. ENSPVAT00000000244,


which is hereby incorporated by reference in its entirety)








1
ATGCCGAGGG GCAGCCTAGG GCTCCTGGTC TGCATCCTGT GCTGCAGAGC CTATCCCGAT


61
GCCTCTCCGC TGCTTAGCTC CAGCTTGGGG GGCCTGATCC ACCTCTACAC AGCCACAGCC


121
AGGAACGGCT ACCACCTGCA GATCCACAAG GATGGCCATG TGGATGGCAC ACCCCATCAG


181
ACCATCTACA GTGCCCTGAT GATAAGATCA GAGGACAGTG GCTTTGTGGT GATAATAGGT


241
GTGATGAGTA GAAGATACCT CTGCATGGAC TTCAAAGGCA ACATTTTTGG ATCACATCAC


301
TTCAGCCCCG AGAGCTGCAA GTTCCGCCAG CGAACGCTGG AGAATGGCTA CGACGTGTAT


361
CACTCGCCCC AGCATCACTT CTTCGTCAGC CTGGGCCGAG CTAAGAGGGC CTTCCTGCCG


421
GGCACGAACC CCCCACCTTA CTCCCAGTTC CTGTCCCGAA GGAATGAGAT CCCCCTGTTC


481
CAGTTCAACA CCCCGCGGCC GCGGCGGCAC ACGCGCAGCG TGGAGGACTA CAAAGACTAC


541
GATTTGGACC CCGACCCGCT GAAAGTTCTG AGGCCCCGTC CCCGGTGGGT CCCCGCCCTG


601
CCCTCCTGCT CCCAGGAGCT CCCGAGTGCC GAGGACAACA GCGTGGTAGC CAACGACCCG


661
TTAGGGGTGC TCAGGCCCAG CAGGGTAAAC ATATACCGTG AGAGAATGGG CAAGGGGAGG


721
TGCCGTCCCC ACCCTGAGTT TGTCTAG


 







Microbat FGF23 gene coding sequence (1-248) (SEQ ID


NO: 301) (Ensembl accession no. ENSMLUT00000031180,


which is hereby incorporated by reference in its entirety)








1
ATGCCAGGGG CCCGCCTTGG GTTGCTGGTC TGCGTCCTGG CCCTGCGCTG TGTGGTCAGA


61
GCCTATCCCA ACGCCTCCCC ACTGCTCGGC TCCAGCTGGG GTGGCCTGAC CCACCTGTAC


121
ACGGCCTCAG CCAGGAACAG CTACCACCTG CAGATCCACA AGGACGGCCA TGTGGACGGC


181
ACACCCCATC AGACCATCTA CAGTGCCCTG ATGATCAGAT CAGAGGACGC TGGCTTTGTG


241
GTGATAACTG GAGTGATGAG TAGGAGATAC CTCTGCATGG ACTTTAGAGG CAACATTTTT


301
GGATCCCTTT TTTTCAGTCC AAGTAATTTC AGTTTCCTTG AATGGAAAAA GGAAAGTGGG


361
ATGGACCATT GGATAAGCAG ACAGACGCAC TTCCTCGTCA GCCCTGGGCC GAGCCAAGAG


421
GGCCTTCCTG CCGGGCACAA CCCGCCGCCC TACTCGCAGT TCCTGTCGCG AAACGAGATC


481
CCGCTCTTCC ACTTCAACAC GCCCGCGCCG CGCCGGCACA CGCGCAGCGC CGAGGAGAAC


541
TCGGCGGCCG ACCCGCTGGT CGTGCTGAAG CCCGTGCCGC GCCTGACGCC CCCGCCCGCC


601
TCCTGCTCCC GGGAGCTGAG CAGCGCCGAG GACAACAGCG TGGCGGCCCA CGACCCGCTC


661
GGGGTGCTGC GGAGCAGCAA CAGGGTGAAC TCGCACGCGC CGCCCCCAGG TCCACCTAGG


721
ACCCGCCAAG GAATGCTTCT CGTA


 







Tasmanian devil FGF23 gene coding sequence (1-245) (SEQ


ID NO: 302) (Ensembl accession no. ENSSHAT00000010240,


which is hereby incorporated by reference in its entirety)








1
ATGTCAGGGG GTTGCCTCAG GCTCCTATTC TGTGCCCTGT GCAGCTTAAG GGCCATCCAA


61
GCCTTCCCCA ATGCTTCCCC CCTGCTCAGC CTTGGCTGGG GGGGTCTGAC TCACCTCTAT


121
ACGGCCACAG CCAGGAACAG CTACCACCTG CAGATCCACA AAGATGGCCA CGTGGATGGG


181
TCTCCTCATC AAACCATCTA TAGTGCCTTG ATGATCAGAT CAGAGGATGC TGGGCTAGTC


241
ATAATAACTG GTGTGATGAG CAGGAGATAT CTCTGTATGG ACATTAGGGG CAACATCTTC


301
GGATCGCATT TCTTCAGCCC AGACAACTGC AGGTTCAAAC ACCGGACATT AGAAAATGGG


361
TATGACATCT ATCACTCTCC CCAGAACAAC TTCCTGATCA GCCTTGGCAA GGCAAAGAGG


421
GCCTTCCTAC CAGGGATGAA CCCACCTCCT TACTCCCAAT TCCTGTCTCG GAGAAATGAA


481
ATCCCCATAA TACACTTCAA TACACCTGAA CCCCACCGGC ATACCAGGAG TGCTGAGAAC


541
AGTCCTGACT TGGACCCAAT GAATGTGCTG AAACTCCGAC CAAGGATAAC TCCCTGCTCC


601
CAGGAACTTC ACAGTGCTGA AGAGAACAGT GTAGTGGATG ATGACCCTTT GGAAGTACTC


661
AGAAATAGCA ATAGATTGAA GCCCTATCCT GGCAGGATGA GTTTGGAAAG ATGCCTCCAT


721
GTCCCCAAGG CAGCTTAA


 







Gray short-tailed opossum FGF23 gene coding sequence


(1-191) (SEQ ID NO: 303) (GenBank accession


no. XM_001372399,


which is hereby incorporated by reference in its entirety)








1
ATGGCAAATT GTAGAGAAAA GGAGCTGGAG ATGTACATTT GTGCCTTGAT GATCAGATCA


61
GAGGATGCTG GGCTAGTCAT AATAACTGGT GTGATGAGCA GGAGATATCT CTGTATGGAC


121
ATCAGGGGCA ACATCTTTGG TTCGCATTTC TTCAACCCGG ACAACTGCAA GTTCAAGCAC


181
CGGACACTAG AAAATGGGTA TGACATCTAT CATTCTCCCC AGAACAACTT CCTGATCAGC


241
CTTGGCAAGG CAAAGAGGGC CTTTCTGCCA GGCATGAATC CACCTCCGTA CTCTCAATTC


301
CTGTCTCGGA AGAATGAGAT CCCCATAATC CACTTCAACA CACCTGAACC CCACCGGCAC


361
ACCAGGAGTG CTGAAAACAG TCCTGACTTG GACCCAATGA ATGTGCTGAA ACCCCGACCA


421
AGGATGACTC CCTGCTCTCA GGAACTCTAC AGTGCTGAAG AGAACAGTGT AGTGGATGAT


481
GACCCTTTGG AAGTACTTAG AAATAGCAAT CGACTGAAGC CCTTCCCTGG TAGGCTGGGT


541
TTAGAAAGGT GCCACCATGT TCCCAAGACT GATTAA


 







Armadillo FGF23 gene coding sequence (1-180) (SEQ


ID NO: 304) (Ensembl accession no. ENSDNOT00000005805,


which is hereby incorporated by reference in its entirety)








1
GCCCTGATGA TCAGCTCTGA AGATGCTGGC TTTGTGGTGA TAACAGGTGT GATGAGCAGG


61
AGGTACCTCT GTATGGATTT CAGAGGCAAC ATTTTTGGAT CGCACGACTT CACCCCGGAC


121
AGCTGCAGGT TCCGCCAGCG CACGCTGGAG AACGGCTACG ACGTCTACCA CTCGCCGCAG


181
CACCACTTCC TCGTCAGCCT GGGGCGGGCC AAGCGGGCCT TCCAGCCGGG CTCCAACCCG


241
CCGCCCTACT CCCAGTTCCT GTCCCGCAGG AACGAGATCC CGCTGATGCG CTTCAGCACC


301
CCGCGGCCGC GGCGGCACAC GCGCAGCGCC CAGGACCACG CGGACCCCGA CCCGCTGAGG


361
GTGCTCAAGC CCCGGCTCCG GCTGACCCCG GCCCCCGCCT CCTGCTCCCA GGAGCTGCCG


421
AGCGACGAGG ACGACGGCGC GGTGGCCAGC GACCCCCTGC GCGTGGTCCT CGGCCGCCGG


481
CCCCACGCGC GGGCCGCGGG CGCGGGCGGG GAGCGGTGCC GCCCCGGCCC GCAGCTCAGC


541
TAG


 







Wallaby FGF23 gene coding sequence (1-177) (SEQ ID


NO: 305) (Ensembl accession no. ENSMEUT00000004101,


which is hereby incorporated by reference in its entirety)








1
GCCTTGATGA TCAGATCAGA GGACGCTGGG CTAGTCATAA TAAGTGGTGT GATGAGCAGG


61
AGGTATCTCT GTATGGACCT CAGAGGCAAC ATCTTCGGAT CGCATTTCTT CAGCCCAGAC


121
AACTGCAGGT TCAAACACCG GACACTAGAA AATGGGTATG ACATCTATCA CTCTCCACAG


181
AACAACCTCC TGATCAGCCT TGGCAAGGCA AAAAGGGCCT TCCTGCCAGG CATGAACCCA


241
CCTCCTTACT CCCAGTTCCT ATCTCGGAGG AATGAGATCC CCATAATCCA CTTCAATACA


301
CCTGAACCCC GCCGGCACAC CAGGAGCGCA GAGAACAGTC CTGACTTGGA CCCAATGAAT


361
GTGCTGAAAC CCCGACCAAG GGTGACTCCC TGCTCCCAGG AACTCCGCAG TGCTGAAGAG


421
AACAGTGTAG TAGATGATGA CCCTTTGGAA GTACTCAGAA ATAGTAATCG CCTGAAGCCC


481
TACCCTGGTA GAATGAGTTT GGAAAGATGC CTCCAAGTCC CCAAAGCTGC TTAA


 







Zebra finch FGF23 gene coding sequence (1-256) (SEQ


ID NO: 306) (GenBank accession no. XM_002190484,


which is hereby incorporated by reference in its entirety)








1
ATGGAGTGGA GAGCCACTCT CCAGGGCATT CCCTGCAGCT CCCTGCTCCT GCTGCTCTGC


61
AGCCTAAAGG CTTCCCTTGC CTTTCCCAAC TCCTCTCCAC TGCTGAGTCC CAGCTGGGGC


121
AATGGAGATC GCCTGATGCA CCTCTACACC GACACCGAGA GGAGCAGCTT CCACCTCCAG


181
ATCAACGCTG ATGGCTACAT CGATGGCGCT CCTCACCAAA CCATCTACAG TGCCCTAATG


241
ATCAAGTCTG AGGGTGCTGG CTCAGTAATA ATCACAGGTG TGAAGAGTGG ACGCTACCTG


301
TGTATGGACA TGAAAGGAAA TATATTTGGC TCGCATTACT TCAGCCAAGA GGACTGCATG


361
TTCAACCACA GGACGCTGGA AAATGGGTAC GATGTGTACC AATCCCCCAA ACACCACTTC


421
TTGGTGAGCT TAGGCAGAGT TAAACAAGTC TTCTCCCCTG GTATGAATCC ACCACCATAC


481
TCCCAGTTTC TGTCCAGGAA GAATGAGATC CCTCTGTTCC GATTCAACAC CCCCGAGCCC


541
CACAGGCACA CCAGGAGTGC AGATGTTGAT CCCGTAGATC CTCACCAGAT CCTGGTCCCG


601
CAGAGGAAGA CCCCAGTGTT TGGCTCCCTG CAGCAGCAGC CAGCAGACTT TCCCCACATG


661
CCCAGGGAGC CCATGAGGAT CAACCAGAAC GACGTGGTGA ACCCCGATGA TCCCCACGCA


721
ATGATGGAGG CCAGGAGGTA CCCAAGCCCC CGCTTCTACA TCACGAGATA A


 







Chicken FGF23 gene coding sequence (1-254) (SEQ ID


NO: 307) (GenBank accession no. XM_425663,


which is hereby incorporated by reference in its entirety)








1
ATGCCACACA CCAGTCCCTG CAGCTGCCTG GAGTACATGC TGCTTGTGCT CTGTATCCTG


61
AAGGCTGCAG TCGCCTTCCC CAACTCCTCT CCGCTGCTGA ATCCCAGCTG GGGGAATGGA


121
GATCAGCTGA TGCACTTGTA CACTTCTACA GAGAGGAACA GCTTCCATCT CCAAATCAAT


181
GCTGATGGAC ACATCAATGG TGTTCCTCAC CAAACCATTT ACAGTGCCTT AATGATCAAG


241
TCTGAGGGTG CTGGCTGTGT AATAATCACA GGTGTGAAGA GTGGACGCTA CCTATGCATG


301
GACATGAAAG GAGACATTTT TGGATCGTAT TATTTCAGCC AAGAGGACTG TGTGTTCAAC


361
CAAAGGACAC TGGAAAATGG ATATGATGTG TACCAATCTC CCAAGCACAA TTTTCTGGTT


421
AGCTTGGGCA GAACTAAGCA AGTTTTCTTC CCTGGTATGA ATCCACCACC ATACTCCCAG


481
TTTTTGTCCA GGAGAAACGA AATCCCTTTG TTTCGATTCA ACACACCTGA ACCCCACAGA


541
AACACTAGAA GTGCAGATGT CGATCCACTG GATCCTCACC AAATCCTGGT CCCACAGAGA


601
AAGGTCTCTG CATTAGGGTC TCAGCTGCAG CTGCAAATGG ACTTTTCCCA TGTGCCCAGA


661
GAACCCATGA GAGTCAATCA GAATGATGTG GTCAATCCAG ATGACCCACA TGCTATGATG


721
GATGCTAGGA GGTATGCTAG TCCTCGCTTT TACATTACAA GATAA


 







Turkey FGF23 gene coding sequence (1-254) (SEQ ID


NO: 308) (GenBank accession no. XM_003202575,


which is hereby incorporated by reference in its entirety)








1
ATGCCGCACA CCAGTCCCTG CAGCTGCCTG GAGTACATGC TGCTTGTGCT CTGTATCCTG


61
AAGGCTGCAG TCAGCTTCCC CAACTCCTCT CCACTGCTGA ATCCCAGCTG GGGGAACGGA


121
GATCAGCTGA TGCACTTGTA TACTTCTACA GAGAGGAACA GCTTCCATCT TCAAATCAAT


181
GCTGATGGCC ACATCAGTGG TGTTCCTTAC CAAACCATTT ACAGTGCCCT AATGATCAAG


241
TCTGAGGGTG CTGGCAGCGT TATAATCACA GGTGTGAAGA GTGGACGCTA CCTATGCATG


301
GACATGAAAG GAGACATTTT TGGATCGCAT TATTTCAGCC AAGAGGACTG CGTGTTCAAC


361
CAAAGAACAC TGGAAAATGG ATATGATGTG TATCAATCTC CCAAGCACAA TTTTCTGGTT


421
AGCTTAGGCA GAACTAAGCA AGTTTTCTTC CCTGGTATGA ATCCACCACC GTACTCCCAG


481
TTTTTGTCCA GGAGAAACGA AATCCCGTTG TTTCGATTCA ACACACCTGA ACCCCACAGA


541
AACACTAGAA GTGCAGATGT TGATCCAATG GATCCTCACC AGATCCTGGT CCCACAGAGA


601
AAGGTCTCTG CAATAGAGTC TCAGCTGCAA CTGCAAATGG ACTTTTCCCA TGTGCCCAGA


661
GAACCCATGA GAGTCAATCA GAACGATGTG GTCAACCCAG ATGACCCACA CGCTATGATG


721
GATGCCAGGA GATATGCTAG TCCTCGCTTT TACATTACAA GATAA


 







Green anole FGF23 gene coding sequence (1-242) (SEQ ID


NO: 309) (GenBank accession no. XM_003221363,


which is hereby incorporated by reference in its entirety)








1
ATGGTCCAGG CTACTCTATA CAGCTTCCTC AAATATATGC TGCTTGCAAC ATGTAGCTGG


61
AAAGCAATTG CTGCTTTCCC CAACGCATCA CCTTTGCTCA GCCTCAACTG GGGAAATTCA


121
GACAGCCTGC TACACTTGTA CACTTCCACA GCAAGAAACA GCTTCCACCT GCAAATCCAC


181
TCCAATGGCT ACGTGGATGG AAGTCCGTAT CAAACAATTT ACAGTGCCTT GATGATCAAA


241
TCTGAAGTTG CTGGTTATGT TATAATAAAT GGTGTGAAAA GTGGACGTTT TCTTTGTATG


301
GATATGAATG GGAACATCTT TGGATCGCAT TTCTTCAGTT ATGAGGACTG CACTTTCAAA


361
CACTGGGTCC TGGAAAATGG TTATGATGTT TATCAGTCTC CCAAATACAA CTACCTTGTC


421
AGCTTAGGAA AAGCAAAGCA ACCATTGTTC CCCAATATGA ATCCACCACC TTACTCCCAG


481
TTCTTGTCCA GGAGAAATGA AATTCCTTTA GTCCAGTTCA ACACACCGAA ACCTCACAGA


541
CATACCAGAA GTGCCAACGC GGATCCCTGC GGCAGCATCA TATCATCAGG AAATATTGCG


601
AAAGAAAACC TACAGTTACA GCCACTAATG TATAACACTA AAATGAATTC AAACAGTGAA


661
GATGAAGACC CAAACAGTGC AATAATCAAT AGAAGATTTT TGAGTCCTAG AACAGATGTC


721
AGGAGCTGA


 







Coelacanth FGF23 gene coding sequence (1-249) (SEQ


ID NO: 310) (Ensembl accession no. ENSLACT00000020646,


which is hereby incorporated by reference in its entirety)








1
CTAGAGTCCG CTCTTCTTGC GTTTTCTATG GCTATATTCT ATAGCTTTAA AGCTGTGAGC


61
TCTTTTCCAA ATTCTTCGCC ACTGCTTAAC CCAGTCTGGG GAAACACTGA CAACCTGATA


121
CACCTGTATA CAGCTTCTGA GACGAACAGC TTCCACTTGC AGATCAACTC CGATGGACAT


181
GTGGATGGTA CTCCACACCA AACCGCTTAC AGTGCACTGC TGATCAAGTC CGAGGAGGCT


241
GGTTCTGTAG TTATCCTGGG GGTGAAGAGT GGACGTTACC TCTGTATGGA TATCAAGGGC


301
AATATTATTG GACTGCATCA CTTCAGCAAG GAAGACTGTA CATTCAAACA AGAGGGCTTG


361
GAAAATGGAT TTGATGTGCT GCGCTCACCT AAGCACAACA TTTTGGTCAG CCTTGACAAG


421
ACTAAACGCT CCTACATCCC GGGTATGAAC CTGCCACCTT ACTCACAGTT TTTATCCCGA


481
CAGAATGAAG TAGCTCTGAT CAACTTCATT AACACACCTG ACATACACAG ACATAGTCGA


541
AATGTTGATG TTGATCCTTC AGACCCCCAT GGGATGATAA TTCAGCCTGA TGTGGGTGTT


601
TCATTTCGTA AGTCTTCATC TCTGTTTTCA GATCTGCCCA GAGACTCCAT GAGAACTAGC


661
CATAATGGTA TGGATATGGT TGATCCTGCT GACCCACATG GAATGTTAGA TTCCAGGAGA


721
AGACCAAGTC CAAGGTTCTT TGCAAGATAG


 







Western clawed frog FGF23 gene coding sequence (1-254)


(SEQ ID NO: 311) (GenBank accession no.


XM_002940305,


which is hereby incorporated by reference in its entirety)








25
                          ATGACC AAGCAGCAAA CTAGACTAGG ACTGGTGCTC


61
ACTGTTCTTG CCAGTATAAA GGTTATATCT GCCTTCCCCA ACTCTTCTCC AATAATCAGT


121
GGCGGCTGGG GGGTCCCTGA CAGACTGATG CACCTATATA CGGCCAGTGA CTGGAACAGC


181
TTCCACCTAC AGATCAACCA TGATGGAAGC ATTGATGGAA CCCCTACCCA AACCATTTAC


241
AGTGCAATAA TGATCAAATC AGAATCCGCT GGGCACGTGG TTATTACTGG GGTGAAGACT


301
AATCGGTACC TGTGCATGGA TAAAAGTGGG AACATTTTTG GATATCACGA CTTCAACCAC


361
GACGACTGCG TTTTTAAGCA CGAGACTCTG GAGAACAACT TTGACGTTTA CCATTCTCCA


421
AAACACAACT TTGTGATCAG CCTCAAGGAG CCCAAGCATC ATTTCCGCCT CGGCATGGAC


481
CTGCCCCCTT ACTCCCAATT CCTGTCCTTG GAGAATGAAA TCCCCATAAC CAGATTCAAT


541
GCTCCAGAGC CGGAAATGAG AATCCCAGAG GGCAACTTTG CTGACCCCAG CGACATCATA


601
AAGAACCCCA GGAACTGGGA CTTTTCGCAG TCTATTCATA ATCCATTTCA GGATGTGTGG


661
TTGCCGTTCC CCAGCGGTTC ATTACCAATC ATTAGAGCTT CCTTGCCAAT TATTCATAAC


721
AATGTGATTA ATACAGATGA CCCTGAAGAA ATTGTAAAAA TGAAGAGATA CAGATATTTC


781
AAGAGGTAG


 







Cat FGF23 gene coding sequence (1-199) (SEQ ID NO: 312)


(Ensembl accession no. ENSFCAT00000000141,


which is hereby incorporated by reference in its entirety)








1
ATGTCAGGGA CCCGCCTTGG GCTCCTGGTC TCTGTCCTGT GCTGGGTAGT CAGAGCCTAT


61
CCTAACACCT CCCCGCTGCT GGGCTCCAGC TGGGGTGGCC TGACCCACCT GTACACGGCC


121
ACAGCCAGGA ACAGCTACCA CCTGCAGATA CACAAGGACG GCCATGTGGA TGGCACACCC


181
CATCAGACCA TCTACAGTGC CCTGATGATC AGATCGGAGG ATGCCGGCTT TGTGGTGATA


241
ACAGGTGTGA TGAGTCAGAG GTACCTCTGT ATGGACTTCA GAGGCAATAT CTTCGGATCG


301
CACCTCTTCA GCCCCGAGAG CTGCAGGTTC CGACAGCGGA CGCTGGAAAA CGGCTACGAC


361
GTGTACCACT CCCCGCAGCA CCGCTTCCTA GTCAGCCTGG GCCCGGCCAA GAGGGCCTTC


421
CTGCCGGGCA CCAACCGCAT GACCCCCGCG CCGGCCTCCT GCTCCCAGGA GCTCCCAAGC


481
GCCGAGGACA GCGGCGTGGT GGCCAGCGAC CCGTTAGGGG TGCTCAGGGG CAACAGGGTG


541
AACGCGCACG CCGGGGGGAT GGGCGTGGAG AGGTGCCGCC CCTTCCCCAA GTTCAACTAG


 







Chinese softshell turtle FGF23 gene coding sequence


(1-250) (SEQ ID NO: 313) (Ensembl accession no.


ENSPSIT00000012816,


which is hereby incorporated by reference in its entirety)








98
                                        ATG TCACAGCCCA GCCAGTGCAG


121
CTGCCTGAAC TTCATGCTGT TCGTGCTATG TAGCTTCAAA GCTATTGCTG CCTTTCCCTT


181
CTTCTCTTCA CTGCTGAATC CCAGCTGGGG GGAAACGGAT AGTTTGATAC ACCTGTACAC


241
AGCTACTGAG AAGAACAGCT TCCATCTGCA GATCAACCCT GATGGTTATG TTGACGGCAC


301
ACCTCACCAA ACCATTTACA GTGCTCTAAT GATCAAATCT GAGGATGCTG GCTATGTGGT


361
GATAAGTGGT GTAAAGAGTG GGCGCTACCT ATGTATGGAC ATTAAAGGAA ATATCTTTGG


421
ATCGCATTAC TTCAGTCAAG AGGACTGCAT GTTTAAACAC AGAACACTGG AAAATGGATA


481
TGATGTGTAC CAGTCTCCCA AGCACAACTT CCTGGTCAGC CTGGGCAGGA ATAAACAAGC


541
TTTCTTCCCT GGTATGAATC TGCCACCATA CTCCCAGTTT TTGCCCAGGA GAAATGAAAT


601
CCCTCTGATC CGATTCAACA CACCCGAACC CCACAGGCAC ACTAGGAATG CAGATGTTGA


661
TCCCCTCCAG ATTTTGATCC CTCGGGGAGA GGCTTTTGAC ACAGGACCTC AGAGGTTGCA


721
GACTCACTTT GATCACCTGC CTAGAGAACC CATGAGAATC AATCCAAATG ATGTAGTCAG


781
CCCGGATGAC CCACTCGCCA TGATGGATGT CAGAAGGAAT GCAAGTCCAC GCCTTTACAT


841
TACAAGA


 







Ferret FGF23 gene coding sequence (1-245) (SEQ ID NO: 314)


(Ensembl accession no. ENSMPUT00000009396,


which is hereby incorporated by reference in its entirety)








186
     ATGTC AGTGACCCGT CTTGGGCTCC TGGTCTCTGT CCTGTGCTGG GTAGTCAGAG


241
CCTATCCCAA CGCCTCCCCG CTGCTCGGCT CCAGCTGGGG TGGCCTGACC CACCTGTACA


301
CGGCCACTGC CAGGAACAGC TACCACCTGC AGATCCACAA GGATGGCCAT GTGGATGGCA


361
CACCCCACCA GACCATCTAC AGCGCCCTGA TGATCAGATC AGAGGATGCC GGCTTTGTGG


421
TGATCACAGG TGTGATGAGC AGGCGGTACC TGTGTATGGA CTTCCGAGGC AACATCTTTG


481
GATCCCACCT CTTCAGCCCC GAGAGCTGCA GGTTCCGACA GCGGACACTG GAAAACGGCT


541
ACGACGTGTA CCACTCCCCG CAGCACCGCT TCCTCGTCAG CCTGGGCCAA GCCAAGAGGG


601
CCTTCCTGCC GGGCACCAAC CCGCCGCCCT ACTCCCAGTT TCTGTCCCGG AGGAATGAGA


661
TCCCCCTCAT CCACTTCAAC ACCCCCAGGC CGCGGCGTCA CACGCGCAGC GCCGAGGACA


721
TGGAGCACGA CCCGTTGAAC GTGCTGAAGC CCCGGCCCCG CATGACCCCG GCCCCGGCCT


781
CCTGCTCCCA GGAGCTCCCG AGCGCCGAGG ACAACAGTGT GGTGGCCAGC GACCCGTTAG


841
GGGTGCTCAG AGGCAACCGG GTGAACGTGC ACGCGGGGGG GATGGGCGTG GACAGGTGCC


901
GCCCCCTCCC CAAGTTCATC TAG


 







Mouse lemur FGF23 gene coding sequence (1-206)


(SEQ ID NO: 315) (Ensembl accession no.


ENSMICT00000004875,


which is hereby incorporated by reference in its entirety)








1
ATGCTGGGGG CCTGCCTCAG GCTCTGGGTC TGTGCCCTGT GCAGTGTCTG CGGCGTGAGC


61
GTCGTCAGAG CCTATCCCAA CGCCTCCCCG CTGCTCGCCT CCAGCTGGGG TGGCCTGATC


121
CACCTGTACA CGGCCACGGC CAGGAACAGC TACCACCTGC AGATCCACAA GGACGGCCAT


181
GTGGACGGCA CACCCCACCA GACCATCTAC AGTGCCTTGA TGATCAGGTC AGAGGATGCT


241
GGCTTTGTGG TGATCACAGG TGTGATGAGC AGAAGATACC TCTGCATGGA TTTCAGAGGC


301
AACATTTTTG GATCACATGT CTTCAGCGCG GAGAGCTGCA GGTTCAGACA GCGGACGCTG


361
GAGAACGGCT TCGACGTGTA CCAGTCCCCT CAGCACCACT TCCTGGTCAG CCTGGGCCGC


421
GCCAAAGGGG CCTTTCCGGC CGGGGCGAAA CCGCCCCCCT TCCCCCAGTT CCTGCCGCGG


481
GGGAACGAGG CTCCCGGGCG CAAAACGCGG GGGCCCGAGG AAAAAGGGGC CCCACACCCT


541
CTCCGCGGGG TGGAAAGCGG GGGCCGGAAA GGCGGGGCCC CGCCTCTCTG TTTGGAGAGG


601
CTCTCCAGAG CCCGAGAG


 







Orangutan FGF23 gene coding sequence (1-251,


excluding 2-22 and 38-71) (SEQ ID NO: 316)


(Ensembl accession no. ENSPPYT00000006110,


which is hereby incorporated by reference in its entirety)








1
ATG------- ---------- ---------- ---------- ---------- ----------


61
------CGCA AT------GA GTCTTTGCCC TGCCTGGTTT TCTCCATAGG T---------


121
---------- ---------- ---------- ---------- ---------- ----------


181
---------- ---------- ---------- GCCCTGATGA TCAGATCAGA GGATGCTGGC


241
TTTGTGGTGA TTACAGGTGT GATGAGCAGA AGATACCTCT GCATGGATTT CAGAGGCAAC


301
ATTTTTGGAT CACACTATTT CAACCCGGAG AACTGCAGGT TCCAACACCA GACGCTGGAA


361
AACGGGTATG ACGTCTACCA CTCTCCTCAG CATCACTTCC TGGTCAGTCT GGGCCGGGTG


421
AAGAGAGCCT TCCTGCCAGG CATG---CCA CCCCCGTACT CCCAGTTCCT GTCCCGGAGG


481
AACGAGATCC CCCTAATTCA CTTCAACACC CCCGTACCAC GGCGGCACAC CCGGAGCGCC


541
GAGGATGACA CGGAGCGGGA CCCCCTGAAA GTGCTGAAGC CCCGGGCCCG GATGACCCCG


601
GCCCCGGCCT CCTGCTCACA GGAGCTCCCG AGCTCCGAGG ACAACAGCCC GATGGCCAGC


661
GACCCATTAG GGGTGGTCAG GGGCGGTCGA GTGAACACGC ACGCTGGGGG AACGGGCCCG


721
GAAGGCTGCC GCCCCTTCCC CAAGTTCATC


 







Shrew FGF23 gene coding sequence (1-251,


excluding 19-27, 71-105, 198-200, and 236-251)


(SEQ ID NO: 317) (Ensembl accession no. ENSSART00000007775,


which is hereby incorporated by reference in its entirety)








1
ATGTGGGGAC TCCGCCTGGG TCTCTTGGTC GGCCTCCTGG GCTGCGTGGA CAGA------


61
GCCTCCCCGA TGCTGGCGTC CAGCTGGGGC GGCCTGACGC ACCTGTACAC GGCCACGGCC


121
AGGAACAGCT ACCACCTCCA GATCCACAAG GACGGCCTGG TCGACGGCTC CCCGCAGCAG


181
ACCGTCTAC- ---------- ---------- ---------- ---------- ----------


241
---------- ---------- ---------- ---------- ---------- ----CACCAT


301
TTCAGCCCGG AGAGCTGCCG CTTCCAGCAG CGCACGCTGG AGAACGGCTA CGACGTGTAC


361
CAGTCCCCGC AGCACCGCTT CCTCGTGAGC CTGGGCCGGC CCAAGCGCGC CTTCCAGCCG


421
GGCGCCAACC CGCCGCCCTA CGCGCAGTTC CTGGCGCGCC GCAACGAGGT GCCCCTGGCG


481
CGCTTCCACA CGCCCGCGCC GCGCCGCCAC ACGCGCAGCG CGCACGACAA CGGCGACGCC


541
GACCCGCTCA ACGTGCTGGC GCCTCGGGCC ---------G CCGCCGCCGC CTCCTGCTCG


601
CACGAGCTGC CCAGCGCCGA GGACAACAGC GTGGTGGCCA GCGACCCGCT GGGCGTCATC


661
CGCAGCAACC GCTTCCGCAC GCAC


 







Tetraodon FGF23 gene coding sequence (1-263) (SEQ


ID NO: 318) (Ensembl accession no. ENSTNIT00000014553,


which is hereby incorporated by reference in its entirety)








1
ATGGACGTAA ACAGAAGGAT CGGGGTGAAG GACGCCTTGC TGGCGCTCCT GCTCGCCCTT


61
CTCCAGGGAT GCCCCCTGGG GGAAACGGCT CCCAACGCGT CACCGCTGGT CGGTTCCAAC


121
TGGGGGAACC CGAGGAGGTA CGTTCACCTT CAGACATCCA CAGACATGAG CAACTTCTAC


181
TTGGAGATCA GACTGGATGG AACCGTGCGC AAAAGCACAG CCCGGACTTC ATACAGTGTG


241
ATTTTACTGA AAGCCGACAC GAGGGAGCGC ATCGCCATCC TGGGCGTCAA GAGCAACCGT


301
TACCTGTGTA TGGACCTCGA GGGGAGCCCA TTTAGCTCTC CCACCTGCAT CAGGGACGAC


361
TGCTTGTTCA ACCACAGTCT TCTGGAGAAC AACCGGGACG TCTACTACTC CAGCCGGACC


421
GGCATTCTCT TCAACCTTGA GGGCTCCCGC CAGGTGTTCG TGGTGGGCCA GAACGTCCCG


481
CAGACCTCCC TCTTCCTGCC CAGGACGAAC ACGGTGCCGC TGGAGCGACT CCTTCTGCAC


541
AGGGACAAGC GGAACCAGGT GGTGGACCCC TCTGACCCGC ACCGCGTCGC CGTGGGTCGC


601
GCCGAGGAGG GCTCGGACTC CCGGGCCTTG CAGGAGGACG ACGCCGACCT GGAGGTGGAG


661
ACAGAGGTTG AGGTCGGGGA CGACGGACGC AACGCGTCCC GGGAGCGGCT GCAGGCTCCG


721
TCCGATCACG ACCCCTGGGG CGTGTTCTCC TCCAACCCCG GGAGCCCCCG CAGCAGCGGC


781
ACGGTGGGCT GA


 







Tilapia FGF23 gene coding sequence (1-255) (SEQ ID


NO: 319) (Ensembl accession no. ENSONIT00000000020,


which is hereby incorporated by reference in its entirety)








472
                                                        ATGGACGTC


481
AACAGGCGAA TGGGGATGAG AGACACCGTG CTGGCGCTCT TTCTCGCTGT CTTGCAGGGA


541
TTTCCTCTCG GGGATACGGT CCCGAACCCA TCACCTCTGG CTGGATCCAA CTGGGGGAAC


601
CCAAGGAGAT ACGTCCACCT GCAGACATCC ACAGACCTCA ATAACTTCTA CTTGGAGATC


661
AGATTAGATG GGAGTGTGCG CAAAACTACG TCCAGGAGCA CCTATAGTGT GATTCTACTG


721
AAATCTGAAG CAAGAGATCG CGTCGCCATC CTCGGCGTCA AAAGCAGCCG TTACCTATGC


781
ATGGACCTGG AGGGCAACCC GTTCAGCTCT CCTGTCTGCC TTCGGGATGA CTGTCTGTTC


841
AACCACAAGC TCCTGGAGAA CAACCGGGAC GTGTACTACT CCAGCCGGAC AGGCATCTTG


901
TTCAACCTGG AGGGCTCCCG ACAGGTGTAC TCGGTGGGCC AGAACCTGCC GCAGACCTCC


961
CTCTTCTTGC CCAGGAAAAA CACCGTACCA CTGGAGCGCC TCCTGCTGCA CAGGGAGAAG


1021
AGAAACCGGG GGCAGACAGA AGAGGGTTCG GACTCCCGGG CCGTGCCGGA GGAGCTGGAG


1081
GAAAGGGAGG TGGAAATGGA GACGGAAATA GAAACAGAGG TCGGGGATGA CGGACGCAAC


1141
GTGTCCCGGG AGAAACTCGC GGCTCCATCC AGCCACGACC CCTGGAACGT GCACTTCTCC


1201
AACCCGGCCA GCCCCCGGAG CACCGGGACA GTGGGCTGA


 







Zebrafish FGF23 gene coding sequence (1-258) (SEQ ID


NO: 320) (Ensembl accession no. ENSDART00000067388,


which is hereby incorporated by reference in its entirety)








79
                   AT GCGTTGCGCA CTTTCCAACC TGCACATGCT GCATTCATCC


121
GTCCTCGCGC TGTGGTTCAC GGCTCTCCAG GGACTCAGAC CTGCAGATGC GGCCCCCAAT


103
CCTTCTCCGC TGCTGGGCTC CAACTGGGGG AACCCGCGGA GATACATCCA CCTTCAGACC


163
ACTTCAGACT TAAACAACTA CTACCTGGAG ATCAGCCCGA GTGGACACGT GCGCAAAACT


223
ACAAATCGGG GCTCATACAG TGTAATCTTA TTGAAAACAG AAAGCAGAGA CCGTCTGGCG


283
ATATTTGGAG TGAAAAGTAA CCGGTTTTTG TGCATGGATA CAGGAGGAAC CCTTTTCACA


343
TCTACGATCT GCAATAAGGA AGACTGTCTT TTCCACCACA AACTGTTGGA AAACCATCGT


403
GATGTGTATT ACTCCACTAA ACACAGCATA CTGCTTAATC TGGACGGGGA CAAACAGGCG


463
TTTATAGCGG GACAAAACCT CCCTCAGTCG TCTCTCTTCT TGTCGGAGAA GAACACGGTT


523
CCGCTGGAGC GCCTGCAGCA TCGGGAGCGC AGGAACCGGC AGGTGAACCC AACAGACCCG


583
CTGAACGCGC TCCGGTACGC GGAGGAGTCT GATTCCAGAG CCGCGCAGGA GGATGATGGA


643
GACATGGATT TTGAGCCCTC AGAAGGTCAA AACATCTCTA GAGAAACCCT TGTTTCCCCT


703
TCCGATGATG ATCCATGGGA TCTTCTGCAC GACACGAGCC CTGGAAGTCC TCGGATTGCA


763
GCAATTGTCG GATAA









Chimeric proteins according to the present invention may be isolated proteins or polypeptides. The isolated chimeric proteins of the present invention may be prepared for use in the above described methods of the present invention using standard methods of synthesis known in the art, including solid phase peptide synthesis (Fmoc or Boc strategies) or solution phase peptide synthesis. Alternatively, peptides of the present invention may be prepared using recombinant expression systems.
In one embodiment, the chimeric protein of the present invention includes the amino acid sequence of SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, or SEQ ID NO: 324, as shown in Table 9.








TABLE 9





Description of Chimeric Protein
Sequence







Amino acid sequence of a
SEQ ID NO: 321


FGF1/FGF23 chimera composed
MAEGEITTFT ALTEKFNLPP GNYKKPKLLY


of residues M1 to L150 of human
CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


FGF1 harboring
LSAESVGEVY IKSTETGQYL AMDTDGLLYG


K127D/K128Q/K133V triple
SQTPNEECLF LERLEENHYN TYISKKHAEK


mutation (bold) and residues
NWFVGLDQNG SCVRGPRTHY GQKAILFLPL


R161 to I251 of human FGF23
RNEIPLIHFN TPIPR HT S AEDDSERDPL


(bold) harboring R176Q/R179Q
NVLKPRARMT PAPASCSQEL PSAEDNSPMA


double mutation (bold italic)
SDPLGVVRGG RVNTHAGGTG PEGCRPFAKF I


 


Amino acid sequence of a
SEQ ID NO: 322


FGF1/FGF23 chimera composed
                          KPKLLY


of residues K25 to L150 of human
CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ


FGF1 harboring
LSAESVGEVY IKSTETGQYL AMDTDGLLYG


K127D/K128Q/K133V triple
SQTPNEECLF LERLEENHYN TYISKKHAEK


mutation (bold) and residues
NWFVGLDQNG SCVRGPRTHY GQKAILFLPL


R161 to I251 of human FGF23
RNEIPLIHFN TPIPR HT S AEDDSERDPL


(bold) harboring R176Q/R179Q
NVLKPRARMT PAPASCSQEL PSAEDNSPMA


double mutation (bold italic)
SDPLGVVRGG RVNTHAGGTG PEGCRPFAKF I


 


Amino acid sequence of a
SEQ ID NO: 323


FGF2/FGF23 chimera composed
MAAGSITTLP ALPEDGGSGA FPPGHFKDPK


of residues M1 to M151 of human
RLYCKNGGFF LRIHPDGRVD GVREKSDPHI


FGF2 harboring
KLQLQAEERG VVSIKGVCAN RYLAMKEDGR


K128D/R129Q/K134V triple
LLASKCVTDE CFFFERLESN NYNTYRSRKY


mutation (bold) and residues
TSWYVALDQT GQYVLGSKTG PGQKAILFLP


R161 to I251 of human FGF23
MRNEIPLIHF NTPIPR HT  SAEDDSERDP


(bold) harboring R176Q/R179Q
LNVLKPRARM TPAPASCSQE LPSAEDNSPM


double mutation (bold italic)
ASDPLGVVRG GRVNTHAGGT GPEGCRPFAK



FI


 


Amino acid sequence of a
SEQ ID NO: 324


FGF2/FGF23 chimera composed
                          HFKDPK


of residues H25 to M151 of
RLYCKNGGFF LRIHPDGRVD GVREKSDPHI


human FGF2 harboring
KLQLQAEERG VVSIKGVCAN RYLAMKEDGR


K128D/R129Q/K134V triple
LLASKCVTDE CFFFERLESN NYNTYRSRKY


mutation (bold) and residues
TSWYVALDQT GQYVLGSKTG PGQKAILFLP


R161 to I251 of human FGF23
MRNEIPLIHF NTPIPR HT  SAEDDSERDP


(bold) harboring R176Q/R179Q
LNVLKPRARM TPAPASCSQE LPSAEDNSPM


double mutation (bold italic)
ASDPLGVVRG GRVNTHAGGT GPEGCRPFAK



FI









Chimeric proteins according to the present invention may be isolated proteins or polypeptides. The isolated chimeric proteins of the present invention may be prepared for use in accordance with the present invention using standard methods of synthesis known in the art, including solid phase peptide synthesis (Fmoc or Boc strategies) or solution phase peptide synthesis. Alternatively, peptides of the present invention may be prepared using recombinant expression systems.
Accordingly, another aspect of the present invention relates to an isolated nucleic acid molecule encoding a chimeric protein according to the present invention. In one embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, or SEQ ID NO: 328 (as shown in Table 10).








TABLE 10





Description of Chimeric Protein
Sequence







Nucleotide sequence of a
SEQ ID NO: 325


FGF1/FGF23 chimera composed
ATGGCTGAAG GGGAAATCAC CACCTTCACA


of residues M1 to L150 of human
GCCCTGACCG AGAAGTTTAA TCTGCCTCCA


FGF1 harboring
GGGAATTACA AGAAGCCCAA ACTCCTCTAC


K127D/K128Q/K133V triple
TGTAGCAACG GGGGCCACTT CCTGAGGATC


mutation (bold) and residues
CTTCCGGATG GCACAGTGGA TGGGACAAGG


R161 to I251 of human FGF23
GACAGGAGCG ACCAGCACAT TCAGCTGCAG


(bold) harboring R176Q/R179Q
CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT


double mutation (bold italic)
ATAAAGAGTA CCGAGACTGG CCAGTACTTG



GCCATGGACA CCGACGGGCT TTTATACGGC



TCACAGACAC CAAATGAGGA ATGTTTGTTC



CTGGAAAGGC TGGAGGAGAA CCATTACAAC



ACCTATATAT CCAAGAAGCA TGCAGAGAAG



AATTGGTTTG TTGGCCTCGA TCAGAATGGG



AGCTGCGTTC GCGGTCCTCG GACTCACTAT



GGCCAGAAAG CAATCTTGTT TCTCCCCCTG



AGGAACGAGA TCCCCCTAAT TCACTTCAAC



ACCCCCATAC CACGG   CA CACC   AGC



GCCGAGGACG ACTCGGAGCG GGACCCCCTG



AACGTGCTGA AGCCCCGGGC CCGGATGACC



CCGGCCCCGG CCTCCTGTTC ACAGGAGCTC



CCGAGCGCCG AGGACAACAG CCCGATGGCC



AGTGACCCAT TAGGGGTGGT CAGGGGCGGT



CGAGTGAACA CGCACGCTGG GGGAACGGGC



CCGGAAGGCT GCCGCCCCTT CGCCAAGTTC



ATC


 


Nucleotide sequence of a
SEQ ID NO: 326


FGF1/FGF23 chimera composed
             AAGCCCAA ACCCCCATAC


of residues K25 to L150 of human
TGTAGCAACG GGGGCCACTT CCTGAGGATC


FGF1 harboring
CTTCCGGATG GCACAGTGGA TGGGACAAGG


K127D/K128Q/K133V triple
GACAGGAGCG ACCAGCACAT TCAGCTGCAG


mutation (bold) and residues
CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT


R161 to I251 of human FGF23
ATAAAGAGTA CCGAGACTGG CCAGTACTTG


(bold) harboring R176Q/R179Q
GCCATGGACA CCGACGGGCT TTTATACGGC


double mutation (bold italic)
TCACAGACAC CAAATGAGGA ATGTTTGTTC



CTGGAAAGGC TGGAGGAGAA CCATTACAAC



ACCTATATAT CCAAGAAGCA TGCAGAGAAG



AATTGGTTTG TTGGCCTCGA TCAGAATGGG



AGCTGCGTTC GCGGTCCTCG GACTCACTAT



GGCCAGAAAG CAATCTTGTT TCTCCCCCTG



AGGAACGAGA TCCCCCTAAT TCACTTCAAC



ACCCCCATAC CACGG   CA CACC   AGC



GCCGAGGACG ACTCGGAGCG GGACCCCCTG



AACGTGCTGA AGCCCCGGGC CCGGATGACC



CCGGCCCCGG CCTCCTGTTC ACAGGAGCTC



CCGAGCGCCG AGGACAACAG CCCGATGGCC



AGTGACCCAT TAGGGGTGGT CAGGGGCGGT



CGAGTGAACA CGCACGCTGG GGGAACGGGC



CCGGAAGGCT GCCGCCCCTT CGCCAAGTTC



ATC


 


Nucleotide sequence of a
SEQ ID NO: 327


FGF2/FGF23 chimera composed
                  ATG GCAGCCGGGA


of residues M1 to M151 of human
GCATCACCAC GCTGCCCGCC TTGCCCGAGG


FGF2 harboring
ATGGCGGCAG CGGCGCCTTC CCGCCCGGCC


K128D/R129Q/K134V triple
ACTTCAAGGA CCCCAAGCGG CTGTACTGCA


mutation (bold) and residues
AAAACGGGGG CTTCTTCCTG CGCATCCACC


R161 to I251 of human FGF23
CCGACGGCCG AGTTGACGGG GTCCGGGAGA


(bold) harboring R176Q/R179Q
AGAGCGACCC TCACATCAAG CTACAACTTC


double mutation (bold italic)
AAGCAGAAGA GAGAGGAGTT GTGTCTATCA



AAGGAGTGTG TGCTAACCGT TACCTGGCTA



TGAAGGAAGA TGGAAGATTA CTGGCTTCTA



AATGTGTTAC GGATGAGTGT TTCTTTTTTG



AACGATTGGA ATCTAATAAC TACAATACTT



ACCGGTCAAG GAAATACACC AGTTGGTATG



TGGCACTGGA TCAGACTGGG CAGTATGTTC



TTGGATCCAA AACAGGACCT GGGCAGAAAG



CTATACTTTT TCTTCCAATG AGGAACGAGA



TCCCCCTAAT TCACTTCAAC ACCCCCATAC



CACGG   CA CACC   AGC GCCGAGGACG



ACTCGGAGCG GGACCCCCTG AACGTGCTGA



AGCCCCGGGC CCGGATGACC CCGGCCCCGG



CCTCCTGTTC ACAGGAGCTC CCGAGCGCCG



AGGACAACAG CCCGATGGCC AGTGACCCAT



TAGGGGTGGT CAGGGGCGGT CGAGTGAACA



CGCACGCTGG GGGAACGGGC CCGGAAGGCT



GCCGCCCCTT CGCCAAGTTC ATC


 


Nucleotide sequence of a
SEQ ID NO: 328


FGF2/FGF23 chimera composed
C


of residues H25 to M151 of
ACTTCAAGGA CCCCAAGCGG CTGTACTGCA


human FGF2 harboring
AAAACGGGGG CTTCTTCCTG CGCATCCACC


K128D/R129Q/K134V triple
CCGACGGCCG AGTTGACGGG GTCCGGGAGA


mutation (bold) and residues
AGAGCGACCC TCACATCAAG CTACAACTTC


R161 to I251 of human FGF23
AAGCAGAAGA GAGAGGAGTT GTGTCTATCA


(bold) harboring R176Q/R179Q
AAGGAGTGTG TGCTAACCGT TACCTGGCTA


double mutation (bold italic)
TGAAGGAAGA TGGAAGATTA CTGGCTTCTA



AATGTGTTAC GGATGAGTGT TTCTTTTTTG



AACGATTGGA ATCTAATAAC TACAATACTT



ACCGGTCAAG GAAATACACC AGTTGGTATG



TGGCACTGGA TCAGACTGGG CAGTATGTTC



TTGGATCCAA AACAGGACCT GGGCAGAAAG



CTATACTTTT TCTTCCAATG AGGAACGAGA



TCCCCCTAAT TCACTTCAAC ACCCCCATAC



CACGG   CA CACC   AGC GCCGAGGACG



ACTCGGAGCG GGACCCCCTG AACGTGCTGA



AGCCCCGGGC CCGGATGACC CCGGCCCCGG



CCTCCTGTTC ACAGGAGCTC CCGAGCGCCG



AGGACAACAG CCCGATGGCC AGTGACCCAT



TAGGGGTGGT CAGGGGCGGT CGAGTGAACA



CGCACGCTGG GGGAACGGGC CCGGAAGGCT



GCCGCCCCTT CGCCAAGTTC ATC









Another aspect of the present invention relates to a nucleic acid construct including a nucleic acid molecule encoding a chimeric protein according to the present invention, a 5′ DNA promoter sequence, and a 3′ terminator sequence. The nucleic acid molecule, the promoter, and the terminator are operatively coupled to permit transcription of the nucleic acid molecule.
Also encompassed are vectors or expression vectors including such nucleic acid molecules and host cells including such nucleic acid molecules. Nucleic acid molecules according to the present invention can be expressed in a host cell, and the encoded polynucleotides isolated, according to techniques that are known in the art.
Generally, the use of recombinant expression systems involves inserting the nucleic acid molecule encoding the amino acid sequence of the desired peptide into an expression system to which the molecule is heterologous (i.e., not normally present). One or more desired nucleic acid molecules encoding a peptide of the invention may be inserted into the vector. When multiple nucleic acid molecules are inserted, the multiple nucleic acid molecules may encode the same or different peptides. The heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5′→3′) orientation relative to the promoter and any other 5′ regulatory molecules, and correct reading frame.
The preparation of the nucleic acid constructs can be carried out using standard cloning procedures well known in the art as described by Joseph Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Harbor 1989). U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in a suitable host cell.
A variety of genetic signals and processing events that control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation) can be incorporated into the nucleic acid construct to maximize protein production. For the purposes of expressing a cloned nucleic acid sequence encoding a desired protein, it is advantageous to use strong promoters to obtain a high level of transcription. Depending upon the host system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV 5, ompF, bla, 1 pp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV 5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene. Common promoters suitable for directing expression in mammalian cells include, without limitation, SV40, MMTV, metallothionein-1, adenovirus E1a, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR.
There are other specific initiation signals required for efficient gene transcription and translation in prokaryotic cells that can be included in the nucleic acid construct to maximize protein production. Depending on the vector system and host utilized, any number of suitable transcription and/or translation elements, including constitutive, inducible, and repressible promoters, as well as minimal 5′ promoter elements, enhancers or leader sequences may be used. For a review on maximizing gene expression see Roberts and Lauer, “Maximizing Gene Expression On a Plasmid Using Recombination In Vitro,” Methods in Enzymology 68:473-82 (1979), which is hereby incorporated by reference in its entirety.
A nucleic acid molecule encoding an isolated protein of the present invention, a promoter molecule of choice, including, without limitation, enhancers, and leader sequences; a suitable 3′ regulatory region to allow transcription in the host, and any additional desired components, such as reporter or marker genes, are cloned into the vector of choice using standard cloning procedures in the art, such as described in Joseph Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Harbor 1989); Frederick M. Ausubel, SHORT PROTOCOLS IN MOLECULAR BIOLOGY (Wiley 1999); and U.S. Pat. No. 4,237,224 to Cohen and Boyer, which are hereby incorporated by reference in their entirety.
Once the nucleic acid molecule encoding the protein has been cloned into an expression vector, it is ready to be incorporated into a host. Recombinant molecules can be introduced into cells, without limitation, via transfection (if the host is a eukaryote), transduction, conjugation, mobilization, or electroporation, lipofection, protoplast fusion, mobilization, or particle bombardment, using standard cloning procedures known in the art, as described by JOSEPH SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Harbor 1989), which is hereby incorporated by reference in its entirety.
A variety of suitable host-vector systems may be utilized to express the recombinant protein or polypeptide. Primarily, the vector system must be compatible with the host used. Host-vector systems include, without limitation, the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria.
Purified proteins may be obtained by several methods readily known in the art, including ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration, and reverse phase chromatography. The protein is preferably produced in purified form (preferably at least about 80% or 85% pure, more preferably at least about 90% or 95% pure) by conventional techniques. Depending on whether the recombinant host cell is made to secrete the protein into growth medium (see U.S. Pat. No. 6,596,509 to Bauer et al., which is hereby incorporated by reference in its entirety), the protein can be isolated and purified by centrifugation (to separate cellular components from supernatant containing the secreted protein) followed by sequential ammonium sulfate precipitation of the supernatant. The fraction containing the protein is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the protein of interest from other proteins. If necessary, the protein fraction may be further purified by HPLC.
Another aspect of the present invention relates to a pharmaceutical composition that includes a chimeric protein according to the present invention and a pharmaceutically acceptable carrier.
“Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
The term “pharmaceutically acceptable” means it is, within the scope of sound medical judgment, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and is commensurate with a reasonable benefit/risk ratio.
In one embodiment, the pharmaceutical composition includes an organotropic targeting agent. In one embodiment, the targeting agent is covalently linked to the chimeric protein via a linker that is cleaved under physiological conditions.
Chimeric and/or modified proteins according to the present invention may also be modified using one or more additional or alternative strategies for prolonging the in vivo half-life of the protein. One such strategy involves the generation of D-peptide chimeric proteins, which consist of unnatural amino acids that are not cleaved by endogenous proteases. Alternatively, the chimeric and/or modified proteins may be fused to a protein partner that confers a longer half-life to the protein upon in vivo administration. Suitable fusion partners include, without limitation, immunoglobulins (e.g., the Fc portion of an IgG), human serum albumin (HAS) (linked directly or by addition of the albumin binding domain of streptococcal protein G), fetuin, or a fragment of any of these. The chimeric and/or modified proteins may also be fused to a macromolecule other than protein that confers a longer half-life to the protein upon in vivo administration. Suitable macromolecules include, without limitation, polyethylene glycols (PEGs). Methods of conjugating proteins or peptides to polymers to enhance stability for therapeutic administration are described in U.S. Pat. No. 5,681,811 to Ekwuribe, which is hereby incorporated by reference in its entirety. Nucleic acid conjugates are described in U.S. Pat. No. 6,528,631 to Cook et al., U.S. Pat. No. 6,335,434 to Guzaev et al., U.S. Pat. No. 6,235,886 to Manoharan et al., U.S. Pat. No. 6,153,737 to Manoharan et al., U.S. Pat. No. 5,214,136 to Lin et al., or U.S. Pat. No. 5,138,045 to Cook et al., which are hereby incorporated by reference in their entirety.
The pharmaceutical composition according to the present invention can be formulated for administration orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
In one particular embodiment of the present invention, the pharmaceutical composition according to the present invention is administered with another hypophosphatemic agent, a phosphate binder, a vitamin D antagonist, an analgesic, and/or an anti-inflammatory agent.
The pharmaceutical composition according to the present invention can be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes. The most suitable route may depend on the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
Another aspect of the present invention relates to a method for treating a subject suffering from a disorder. This method involves selecting a subject suffering from the disorder and administering the pharmaceutical composition according to the present invention to the selected subject under conditions effective to treat the disorder. In one embodiment, the disorder is associated with hyperphosphatemia, abnormally high renal phosphate reabsorption, abnormally low blood levels of full-length, bioactive FGF23, inappropriately normal blood levels of bioactive vitamin D, and/or elevated blood levels of bioactive vitamin D. In one embodiment, the disorder is associated with soft tissue calcification.
Accordingly, another aspect of the present invention relates to a method for treating a subject suffering from a disorder. This method involves selecting a subject suffering from the disorder. The method also involves providing a chimeric FGF protein, where the chimeric FGF protein includes an N-terminus coupled to a C-terminus. The N-terminus includes a portion of a paracrine FGF and the C-terminus includes a C-terminal portion of FGF23. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves administering a therapeutically effective amount of the chimeric FGF protein to the selected subject under conditions effective to treat the disorder.
Suitable chimeric proteins for use in accordance with this aspect of the present invention are described above and throughout the present application.
In one embodiment, the selected subject is a mammal. In one embodiment, the selected subject is a human. In another embodiment, the selected subject is a rodent.
In one embodiment, the chimeric FGF protein is effective for treating disorders associated with hyperphosphatemia, abnormally high renal phosphate reabsorption, abnormally low blood levels of full-length, bioactive FGF23, inappropriately normal blood levels of bioactive vitamin D, and/or elevated blood levels of bioactive vitamin D. In one embodiment, the chimeric FGF protein is effective for treating disorders associated with soft tissue calcification. In one embodiment, the chimeric FGF protein normalizes vitamin D metabolism and/or phosphate metabolism. In one embodiment, the chimeric FGF protein ameliorates soft tissue calcification.
The chimeric protein of the present invention or pharmaceutical composition thereof can be used to treat a number of conditions. In one embodiment, the condition is one which the therapeutic outcome includes a decrease in circulating phosphate levels. In one embodiment, the condition is one which the therapeutic outcome includes a decrease in circulating levels of bioactive vitamin D. Each of these parameters can be measured by standard methods, for example, by performing blood tests for phosphate and vitamin D.
In one embodiment, the disorder is associated with hyperphosphatemia, abnormally high renal phosphate reabsorption, abnormally low blood levels of full-length, bioactive FGF23, inappropriately normal blood levels of bioactive vitamin D, and/or elevated blood levels of bioactive vitamin D. In one embodiment, the disorder is associated with soft tissue calcification. In one embodiment, the disorder is tumoral calcinosis (also referred to as hyperphosphatemic familial tumoral calcinosis, Online Mendelian Inheritance in Man, ID 211900 (purl.bioontology.org/ontology/OMIM/211900), which is hereby incorporated by reference in its entirety). In one embodiment, the disorder is associated with hyperostosis, diaphysitis, arterial aneurysms, dental abnormalities, and/or angioid streaks of the retina.
Familial tumoral calcinosis is an autosomal recessive metabolic disorder associated with hyperphosphatemia and soft tissue calcification. Missense mutations in either the UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylglactosaminyltransferase 3 (GALNT3) gene (Garringer et al., “Two Novel GALNT3 Mutations in Familial Tumoral Calcinosis,” Am J Med Genet A 143A:2390-2396 (2007)) or the FGF23 gene (Garringer et al., “Molecular Genetic and Biochemical Analyses of FGF23 Mutations in Familial Tumoral Calcinosis,” Am J Physiol Endocrinol Metab 295:E929-E937 (2008); Araya et al., “A Novel Mutation in Fibroblast Growth Factor 23 Gene as a Cause of Tumoral Calcinosis,” J Clin Endocrinol Metab 90:5523-5527 (2005), each of which is hereby incorporated by reference in its entirety) have been associated with familial tumoral calcinosis. All patients with familial tumoral calcinosis have abnormally high plasma levels of the C-terminal proteolytic fragment of FGF23 but abnormally low plasma levels of intact, full-length FGF23. The excess C-terminal FGF23 fragment may aggravate hyperphosphatemia, and the resulting soft tissue calcification, by antagonizing the action of any residual, functional FGF23 ligand in these patients. Thus, the chimeric and modified proteins according to the present invention are FGF23 agonists that provide a causative form of treatment for these patients.
In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof is administered with a pharmaceutically-acceptable carrier.
The chimeric protein according to the present invention or pharmaceutical composition thereof can be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes. The most suitable route may depend on the condition and disorder of the recipient. Formulations including chimeric proteins according to the present invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Those skilled in the art can readily optimize pharmaceutically effective dosages and administration regimens for therapeutic compositions including the chimeric protein according to the present invention, as determined by good medical practice and the clinical condition of the individual patient.
When in vivo administration of a chimeric protein of the present invention or is employed, normal dosage amounts may vary from, for example, about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day. In one embodiment, the dosage may be from about 1 μg/kg/day to 10 mg/kg/day, depending upon the route of administration. In one embodiment, the chimeric protein according to the present invention is administered at a dose of about 0.1 to 10 mg/kg once or twice daily. In one embodiment, the chimeric protein according to the present invention is administered at a dose of about 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 mg/kg. Guidance as to particular dosages and methods of delivery of proteins is provided in the literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212, which are hereby incorporated by reference in their entirety. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue.
Where sustained-release administration of a chimeric protein of the present invention is desired in a formulation with release characteristics suitable for the treatment of any disease or disorder requiring administration of the chimeric protein of the present invention, microencapsulation is contemplated. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., “Preparation and Characterization of Poly(D,L-lactide-co-glycolide) Microspheres for Controlled Release of Human Growth Hormone,” Nat. Med. 2:795-799 (1996); Yasuda, “Sustained Release Formulation of Interferon,” Biomed. Ther. 27:1221-1223 (1993); Hora et al., “Controlled Release of Interleukin-2 from Biodegradable Microspheres,” Nat. Biotechnol. 8:755-758 (1990); Cleland, “Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems,” in VACCINE DESIGN: THE SUBUNIT AND ADJUVANT APPROACH 439-462 (Powell and Newman, eds. 1995); WO 97/03692; WO 96/40072; WO 96/07399; and U.S. Pat. No. 5,654,010, which are hereby incorporated by reference in their entirety. The sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids, can be cleared quickly within the human body. Moreover, the degradability of this polymer can be adjusted from months to years depending on its molecular weight and composition. Lewis, “Controlled release of bioactive agents from lactide/glycolide polymer,” in: BIODEGRADABLE POLYMERS AS DRUG DELIVERY SYSTEMS 1-41 (M. Chasin and R. Langer eds. 1990), which is hereby incorporated by reference in its entirety.
The chimeric protein of the present invention or pharmaceutical composition thereof may be administered as frequently as necessary in order to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. For other patients, it may be necessary to have long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each particular patient. For other patients, it will be necessary to prescribe not more than one or two doses per day.
In some embodiments, the chimeric protein of the present invention or a pharmaceutical composition thereof is administered in a therapeutically effective amount in combination with a therapeutically effective amount of a second agent. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof is administered in conjunction with the second agent, i.e., the respective periods of administration are part of a single administrative regimen. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof and the second agent are administered concurrently, i.e., the respective periods of administration overlap each other. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof and the second agent are administered non-concurrently, i.e., the respective periods of administration do not overlap each other. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof and the second agent are administered sequentially, i.e., the chimeric protein of the present invention or pharmaceutical composition thereof is administered prior to and/or after the administration of the second agent. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof and the second agent are administered simultaneously as separate compositions. In one embodiment, the chimeric protein of the present invention or pharmaceutical composition thereof and the second agent are administered simultaneously as part of the same compositions.
In one embodiment, the second agent is a hypophosphatemic agent, a phosphate binder, a vitamin D antagonist, an analgesic, and/or an anti-inflammatory agent.
Another aspect of the present invention relates to a method of making a chimeric FGF protein possessing enhanced endocrine activity. This method involves introducing one or more modifications to an FGF protein, where the modification decreases the affinity of the FGF protein for heparin and/or heparan sulfate and coupling a C-terminal portion of FGF23 that includes a α-Klotho-FGFR complex binding domain to the modified FGF protein's C-terminus, whereby a chimeric FGF protein possessing enhanced endocrine activity is made.
Suitable C-terminal portions of FGF23 are described above. In one embodiment, the C-terminal region from FGF23 is derived from a mammalian FGF23. In one embodiment, the C-terminal region derived from FGF23 is from a vertebrate FGF23.
In one embodiment, the chimeric FGF protein has greater binding affinity for FGFR than native FGF23. In one embodiment the chimeric FGF protein possesses enhanced endocrine activity compared to the chimeric FGF protein in the absence of the modification or the α-Klotho-FGFR complex binding domain. In one embodiment, the native endocrine FGF ligand having the α-Klotho co-receptor-FGFR binding domain is native FGF23. In one embodiment, the FGFR is FGFR1c, FGFR3c, or FGFR4.
In one embodiment, the chimeric FGF protein has greater stability than a native endocrine FGF ligand possessing the α-Klotho-FGFR complex binding domain. In one embodiment, increasing the stability includes an increase in thermal stability of the protein as compared to either wild type protein or native endocrine FGF ligand. In one embodiment, increasing the stability includes increasing the half-life of the protein in the blood circulation as compared to wild type or native protein or native endocrine FGF ligand.
In one embodiment, the FGF is derived from a mammalian FGF. In one embodiment, the FGF is derived from a vertebrate FGF. In one embodiment, the FGF protein is a paracrine FGF molecule. In one embodiment the FGF molecule is FGF1 or FGF2. In one embodiment, the FGF protein is an FGF protein that possesses intrinsically greater binding affinity for FGF receptor than a native endocrine FGF ligand. In one embodiment, the FGF protein is an FGF protein that possesses intrinsically greater thermal stability than a native endocrine FGF ligand. In one embodiment, the method involves introducing one or more modifications to the FGF protein, where the modification alters receptor-binding specificity and/or receptor-binding affinity of the FGF protein. In one embodiment, the method involves introducing one or more modifications to the FGF protein, where the modification alters the stability of the FGF protein. For example, receptor-binding specificity of FGF1, which by nature binds to all the seven principal FGFRs, may be altered to, for example, reduce any risk for adverse effects (e.g., mitogenicity). Paracrine FGFs, portions of paracrine FGFs, and modifications thereto are described above.
In one embodiment, the chimeric FGF protein normalizes vitamin D metabolism and/or phosphate metabolism.
Suitable methods of generating chimeric proteins according to the present invention include standard methods of synthesis known in the art, as described above.
Yet another aspect of the present invention relates to a method of facilitating fibroblast growth factor receptor (“FGFR”)-α-Klotho co-receptor complex formation. This method involves providing a cell that includes a α-Klotho co-receptor and an FGFR and providing a chimeric FGF protein. The chimeric FGF protein includes a C-terminal portion of FGF23 and a portion of a paracrine FGF, where the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves contacting the cell and the chimeric FGF protein under conditions effective to cause FGFR-αKlotho co-receptor complex formation.
The portion of the paracrine FGF may also be modified to alter receptor-binding specificity and/or receptor-binding affinity of the FGF, as noted above. Suitable portions of the paracrine FGFs for use in accordance with the present invention, as well as modifications to receptor-binding specificity and/or receptor-binding affinity of the FGF are described above. Suitable modifications to the paracrine FGFs for use in accordance with the present invention are also described above. Suitable C-terminal portions from FGF23 are described above and throughout the present application.
In one embodiment according to the present invention, α-Klotho is mammalian αKlotho. In one embodiment, α-Klotho is human or mouse α-Klotho. In one particular embodiment of the present invention, α-Klotho is human or mouse αKlotho having the amino acid sequence of SEQ ID NO: 329 (i.e., GenBank Accession No. NP_004786, which is hereby incorporated by reference in its entirety) or SEQ ID NO: 331 (i.e., GenBank Accession No. NP_038851, which is hereby incorporated by reference in its entirety), respectively, as follows:








SEQ ID NO: 329:










1
MPASAPPRRP RPPPPSLSLL LVLLGLGGRR LRAEPGDGAQ TWARFSRPPA PEAAGLFQGT



 


61
FPDGFLWAVG SAAYQTEGGW QQHGKGASIW DTFTHHPLAP PGDSRNASLP LGAPSPLQPA


 


121
TGDVASDSYN NVFRDTEALR ELGVTHYRFS ISWARVLPNG SAGVPNREGL RYYRRLLERL


 


181
RELGVQPVVT LYHWDLPQRL QDAYGGWANR ALADHFRDYA ELCFRHFGGQ VKYWITIDNP


 


241
YVVAWHGYAT GRLAPGIRGS PRLGYLVAHN LLLAHAKVWH LYNTSFRPTQ GGQVSIALSS


 


301
HWINPRRMTD HSIKECQKSL DFVLGWFAKP VFIDGDYPES MKNNLSSILP DFTESEKKFI


 


361
KGTADFFALC FGPTLSFQLL DPHMKFRQLE SPNLRQLLSW IDLEFNHPQI FIVENGWFVS


 


421
GTTKRDDAKY MYYLKKFIME TLKAIKLDGV DVIGYTAWSL MDGFEWHRGY SIRRGLFYVD


 


481
FLSQDKMLLP KSSALFYQKL IEKNGFPPLP ENQPLEGTFP CDFAWGVVDN YIQVDTTLSQ


 


541
FTDLNVYLWD VHHSKRLIKV DGVVTKKRKS YCVDFAAIQP QIALLQEMHV THFRFSLDWA


 


601
LILPLGNQSQ VNHTILQYYR CMASELVRVN ITPVVALWQP MAPNQGLPRL LARQGAWENP


 


661
YTALAFAEYA RLCFQELGHH VKLWITMNEP YTRNMTYSAG HNLLKAHALA WHVYNEKFRH


 


721
AQNGKISIAL QADWIEPACP FSQKDKEVAE RVLEFDIGWL AEPIFGSGDY PWVMRDWLNQ


 


781
RNNFLLPYFT EDEKKLIQGT FDFLALSHYT TILVDSEKED PIKYNDYLEV QEMTDITWLN


 


841
SPSQVAVVPW GLRKVLNWLK FKYGDLPMYI ISNGIDDGLH AEDDQLRVYY MQNYINEALK


 


901
AHILDGINLC GYFAYSFNDR TAPRFGLYRY AADQFEPKAS MKHYRKIIDS NGFPGPETLE


 


961
RFCPEEFTVC TECSFFHTRK SLLAFIAFLF FASIISLSLI FYYSKKGRRS YK


 








SEQ ID NO: 331:










1
MLARAPPRRP PRLVLLRLLL LHLLLLALRA RCLSAEPGQG AQTWARFARA PAPEAAGLLH



 


61
DTFPDGFLWA VGSAAYQTEG GWRQHGKGAS IWDTFTHHSG AAPSDSPIVV APSGAPSPPL


 


121
SSTGDVASDS YNNVYRDTEG LRELGVTHYR FSISWARVLP NGTAGTPNRE GLRYYRRLLE


 


181
RLRELGVQPV VTLYHWDLPQ RLQDTYGGWA NRALADHFRD YAELCFRHFG GQVKYWITID


 


241
NPYVVAWHGY ATGRLAPGVR GSSRLGYLVA HNLLLAHAKV WHLYNTSFRP TQGGRVSIAL


 


301
SSHWINPRRM TDYNIRECQK SLDFVLGWFA KPIFIDGDYP ESMKNNLSSL LPDFTESEKR


 


361
LIRGTADFFA LSFGPTLSFQ LLDPNMKFRQ LESPNLRQLL SWIDLEYNHP PIFIVENGWF


 


421
VSGTTKRDDA KYMYYLKKFI METLKAIRLD GVDVIGYTAW SLMDGFEWHR GYSIRRGLFY


 


481
VDFLSQDKEL LPKSSALFYQ KLIEDNGFPP LPENQPLEGT FPCDFAWGVV DNYVQVDTTL


 


541
SQFTDPNVYL WDVHHSKRLI KVDGVVAKKR KPYCVDFSAI RPQITLLREM RVTHFRFSLD


 


601
WALILPLGNQ TQVNHTVLHF YRCMISELVH ANITPVVALW QPAAPHQGLP HALAKHGAWE


 


661
NPHTALAFAD YANLCFKELG HWVNLWITMN EPNTRNMTYR AGHHLLRAHA LAWHLYDDKF


 


721
RAAQKGKISI ALQADWIEPA CPFSQNDKEV AERVLEFDIG WLAEPIFGSG DYPRVMRDWL


 


781
NQKNNFLLPY FTEDEKKLVR GSFDFLAVSH YTTILVDWEK EDPMKYNDYL EVQEMTDITW


 


841
LNSPSQVAVV PWGLRKVLNW LRFKYGDLPM YVTANGIDDD PHAEQDSLRI YYIKNYVNEA


 


901
LKAYVLDDIN LCGYFAYSLS DRSAPKSGFY RYAANQFEPK PSMKHYRKII DSNGFLGSGT


 


961
LGRFCPEEYT VCTECGFFQT RKSLLVFISF LVFTFIISLA LIFHYSKKGQ RSYK






In one particular embodiment of the present invention, α-Klotho is human or mouse α-Klotho encoded by a nucleotide sequence having the nucleotide sequences of SEQ ID NO: 330 (GenBank Accession No. NM_04795, which is hereby incorporated by reference in its entirety) and SEQ ID NO: 332 (GenBank Accession No. NM_013823, which is hereby incorporated by reference in its entirety), as follows:








SEQ ID NO: 330 (human αKlotho gene coding sequence):










9
        AT GCCCGCCAGC GCCCCGCCGC GCCGCCCGCG GCCGCCGCCG CCGTCGCTGT



 


61
CGCTGCTGCT GGTGCTGCTG GGCCTGGGCG GCCGCCGCCT GCGTGCGGAG CCGGGCGACG


 


121
GCGCGCAGAC CTGGGCCCGT TTCTCGCGGC CTCCTGCCCC CGAGGCCGCG GGCCTCTTCC


 


181
AGGGCACCTT CCCCGACGGC TTCCTCTGGG CCGTGGGCAG CGCCGCCTAC CAGACCGAGG


 


241
GCGGCTGGCA GCAGCACGGC AAGGGTGCGT CCATCTGGGA TACGTTCACC CACCACCCCC


 


301
TGGCACCCCC GGGAGACTCC CGGAACGCCA GTCTGCCGTT GGGCGCCCCG TCGCCGCTGC


 


361
AGCCCGCCAC CGGGGACGTA GCCAGCGACA GCTACAACAA CGTCTTCCGC GACACGGAGG


 


421
CGCTGCGCGA GCTCGGGGTC ACTCACTACC GCTTCTCCAT CTCGTGGGCG CGAGTGCTCC


 


481
CCAATGGCAG CGCGGGCGTC CCCAACCGCG AGGGGCTGCG CTACTACCGG CGCCTGCTGG


 


541
AGCGGCTGCG GGAGCTGGGC GTGCAGCCCG TGGTCACCCT GTACCACTGG GACCTGCCCC


 


601
AGCGCCTGCA GGACGCCTAC GGCGGCTGGG CCAACCGCGC CCTGGCCGAC CACTTCAGGG


 


661
ATTACGCGGA GCTCTGCTTC CGCCACTTCG GCGGTCAGGT CAAGTACTGG ATCACCATCG


 


721
ACAACCCCTA CGTGGTGGCC TGGCACGGCT ACGCCACCGG GCGCCTGGCC CCCGGCATCC


 


781
GGGGCAGCCC GCGGCTCGGG TACCTGGTGG CGCACAACCT CCTCCTGGCT CATGCCAAAG


 


841
TCTGGCATCT CTACAATACT TCTTTCCGTC CCACTCAGGG AGGTCAGGTG TCCATTGCCC


 


901
TAAGCTCTCA CTGGATCAAT CCTCGAAGAA TGACCGACCA CAGCATCAAA GAATGTCAAA


 


961
AATCTCTGGA CTTTGTACTA GGTTGGTTTG CCAAACCCGT ATTTATTGAT GGTGACTATC


 


1021.
CCGAGAGCAT GAAGAATAAC CTTTCATCTA TTCTGCCTGA TTTTACTGAA TCTGAGAAAA


 


1081
AGTTCATCAA AGGAACTGCT GACTTTTTTG CTCTTTGCTT TGGACCCACC TTGAGTTTTC


 


1141
AACTTTTGGA CCCTCACATG AAGTTCCGCC AATTGGAATC TCCCAACCTG AGGCAACTGC


 


1201
TTTCCTGGAT TGACCTTGAA TTTAACCATC CTCAAATATT TATTGTGGAA AATGGCTGGT


 


1261
TTGTCTCAGG GACCACCAAG AGAGATGATG CCAAATATAT GTATTACCTC AAAAAGTTCA


 


1321
TCATGGAAAC CTTAAAAGCC ATCAAGCTGG ATGGGGTGGA TGTCATCGGG TATACCGCAT


 


1381
GGTCCCTCAT GGATGGTTTC GAGTGGCACA GAGGTTACAG CATCAGGCGT GGACTCTTCT


 


1441
ATGTTGACTT TCTAAGCCAG GACAAGATGT TGTTGCCAAA GTCTTCAGCC TTGTTCTACC


 


1501
AAAAGCTGAT AGAGAAAAAT GGCTTCCCTC CTTTACCTGA AAATCAGCCC CTAGAAGGGA


 


1561
CATTTCCCTG TGACTTTGCT TGGGGAGTTG TTGACAACTA CATTCAAGTA GATACCACTC


 


1621
TGTCTCAGTT TACCGACCTG AATGTTTACC TGTGGGATGT CCACCACAGT AAAAGGCTTA


 


1681
TTAAAGTGGA TGGGGTTGTG ACCAAGAAGA GGAAATCCTA CTGTGTTGAC TTTGCTGCCA


 


1741
TCCAGCCCCA GATCGCTTTA CTCCAGGAAA TGCACGTTAC ACATTTTCGC TTCTCCCTGG


 


1801
ACTGGGCCCT GATTCTCCCT CTGGGTAACC AGTCCCAGGT GAACCACACC ATCCTGCAGT


 


1861
ACTATCGCTG CATGGCCAGC GAGCTTGTCC GTGTCAACAT CACCCCAGTG GTGGCCCTGT


 


1921
GGCAGCCTAT GGCCCCGAAC CAAGGACTGC CGCGCCTCCT GGCCAGGCAG GGCGCCTGGG


 


1981
AGAACCCCTA CACTGCCCTG GCCTTTGCAG AGTATGCCCG ACTGTGCTTT CAAGAGCTCG


 


2041
GCCATCACGT CAAGCTTTGG ATAACGATGA ATGAGCCGTA TACAAGGAAT ATGACATACA


 


2101
GTGCTGGCCA CAACCTTCTG AAGGCCCATG CCCTGGCTTG GCATGTGTAC AATGAAAAGT


 


2161
TTAGGCATGC TCAGAATGGG AAAATATCCA TAGCCTTGCA GGCTGATTGG ATAGAACCTG


 


2221
CCTGCCCTTT CTCCCAAAAG GACAAAGAGG TGGCTGAGAG AGTTTTGGAA TTTGACATTG


 


2281
GCTGGCTGGC TGAGCCCATT TTCGGCTCTG GAGATTATCC ATGGGTGATG AGGGACTGGC


 


2341
TGAACCAAAG AAACAATTTT CTTCTTCCTT ATTTCACTGA AGATGAAAAA AAGCTAATCC


 


2401
AGGGTACCTT TGACTTTTTG GCTTTAAGCC ATTATACCAC CATCCTTGTA GACTCAGAAA


 


2461
AAGAAGATCC AATAAAATAC AATGATTACC TAGAAGTGCA AGAAATGACC GACATCACGT


 


2521
GGCTCAACTC CCCCAGTCAG GTGGCGGTAG TGCCCTGGGG GTTGCGCAAA GTGCTGAACT


 


2581
GGCTGAAGTT CAAGTACGGA GACCTCCCCA TGTACATAAT ATCCAATGGA ATCGATGACG


 


2641
GGCTGCATGC TGAGGACGAC CAGCTGAGGG TGTATTATAT GCAGAATTAC ATAAACGAAG


 


2701
CTCTCAAAGC CCACATACTG GATGGTATCA ATCTTTGCGG ATACTTTGCT TATTCGTTTA


 


2761
ACGACCGCAC AGCTCCGAGG TTTGGCCTCT ATCGTTATGC TGCAGATCAG TTTGAGCCCA


 


2821
AGGCATCCAT GAAACATTAC AGGAAAATTA TTGACAGCAA TGGTTTCCCG GGCCCAGAAA


 


2881
CTCTGGAAAG ATTTTGTCCA GAAGAATTCA CCGTGTGTAC TGAGTGCAGT TTTTTTCACA


 


2941
CCCGAAAGTC TTTACTGGCT TTCATAGCTT TTCTATTTTT TGCTTCTATT ATTTCTCTCT


 


3001
CCCTTATATT TTACTACTCG AAGAAAGGCA GAAGAAGTTA CAAATAG


 








SEQ ID NO: 332 (murine αKlotho gene coding sequence):










111
                                                       ATGCTAGCCC



 


121
GCGCCCCTCC TCGCCGCCCG CCGCGGCTGG TGCTGCTCCG TTTGCTGTTG CTGCATCTGC


 


181
TGCTGCTCGC CCTGCGCGCC CGCTGCCTGA GCGCTGAGCC GGGTCAGGGC GCGCAGACCT


 


241
GGGCTCGCTT CGCGCGCGCT CCTGCCCCAG AGGCCGCTGG CCTCCTCCAC GACACCTTCC


 


301
CCGACGGTTT CCTCTGGGCG GTAGGCAGCG CCGCCTATCA GACCGAGGGC GGCTGGCGAC


 


361
AGCACGGCAA AGGCGCGTCC ATCTGGGACA CTTTCACCCA TCACTCTGGG GCGGCCCCGT


 


421
CCGACTCCCC GATCGTCGTG GCGCCGTCGG GTGCCCCGTC GCCTCCCCTG TCCTCCACTG


 


481
GAGATGTGGC CAGCGATAGT TACAACAACG TCTACCGCGA CACAGAGGGG CTGCGCGAAC


 


541
TGGGGGTCAC CCACTACCGC TTCTCCATAT CGTGGGCGCG GGTGCTCCCC AATGGCACCG


 


601
CGGGCACTCC CAACCGCGAG GGGCTGCGCT ACTACCGGCG GCTGCTGGAG CGGCTGCGGG


 


661
AGCTGGGCGT GCAGCCGGTG GTTACCCTGT ACCATTGGGA CCTGCCACAG CGCCTGCAGG


 


721
ACACCTATGG CGGATGGGCC AATCGCGCCC TGGCCGACCA TTTCAGGGAT TATGCCGAGC


 


781
TCTGCTTCCG CCACTTCGGT GGTCAGGTCA AGTACTGGAT CACCATTGAC AACCCCTACG


 


841
TGGTGGCCTG GCACGGGTAT GCCACCGGGC GCCTGGCCCC GGGCGTGAGG GGCAGCTCCA


 


901
GGCTCGGGTA CCTGGTTGCC CACAACCTAC TTTTGGCTCA TGCCAAAGTC TGGCATCTCT


 


961
ACAACACCTC TTTCCGCCCC ACACAGGGAG GCCGGGTGTC TATCGCCTTA AGCTCCCATT


 


1021
GGATCAATCC TCGAAGAATG ACTGACTATA ATATCAGAGA ATGCCAGAAG TCTCTTGACT


 


1081
TTGTGCTAGG CTGGTTTGCC AAACCCATAT TTATTGATGG CGACTACCCA GAGAGTATGA


 


1141
AGAACAACCT CTCGTCTCTT CTGCCTGATT TTACTGAATC TGAGAAGAGG CTCATCAGAG


 


1201
GAACTGCTGA CTTTTTTGCT CTCTCCTTCG GACCAACCTT GAGCTTTCAG CTATTGGACC


 


1261
CTAACATGAA GTTCCGCCAA TTGGAGTCTC CCAACCTGAG GCAGCTTCTG TCTTGGATAG


 


1321
ATCTGGAATA TAACCACCCT CCAATATTTA TTGTGGAAAA TGGCTGGTTT GTCTCGGGAA


 


1381
CCACCAAAAG GGATGATGCC AAATATATGT ATTATCTCAA GAAGTTCATA ATGGAAACCT


 


1441
TAAAAGCAAT CAGACTGGAT GGGGTCGACG TCATTGGGTA CACCGCGTGG TCGCTCATGG


 


1501
ACGGTTTCGA GTGGCATAGG GGCTACAGCA TCCGGCGAGG ACTCTTCTAC GTTGACTTTC


 


1561
TGAGTCAGGA CAAGGAGCTG TTGCCAAAGT CTTCGGCCTT GTTCTACCAA AAGCTGATAG


 


1621
AGGACAATGG CTTTCCTCCT TTACCTGAAA ACCAGCCCCT TGAAGGGACA TTTCCCTGTG


 


1681
ACTTTGCTTG GGGAGTTGTT GACAACTACG TTCAAGTGGA CACTACTCTC TCTCAGTTTA


 


1741
CTGACCCGAA TGTCTATCTG TGGGATGTGC ATCACAGTAA GAGGCTTATT AAAGTAGACG


 


1801
GGGTTGTAGC CAAGAAGAGA AAACCTTACT GTGTTGATTT CTCTGCCATC CGGCCTCAGA


 


1861
TAACCTTACT TCGAGAAATG CGGGTCACCC ACTTTCGCTT CTCCCTGGAC TGGGCCCTGA


 


1921
TCTTGCCTCT GGGTAACCAG ACCCAAGTGA ACCACACGGT TCTGCACTTC TACCGCTGCA


 


1981
TGATCAGCGA GCTGGTGCAC GCCAACATCA CTCCAGTGGT GGCCCTGTGG CAGCCAGCAG


 


2041
CCCCGCACCA AGGCCTGCCA CATGCCCTTG CAAAACATGG GGCCTGGGAG AACCCGCACA


 


2101
CTGCTCTGGC GTTTGCAGAC TACGCAAACC TGTGTTTTAA AGAGTTGGGT CACTGGGTCA


 


2161
ATCTCTGGAT CACCATGAAC GAGCCAAACA CACGGAACAT GACCTATCGT GCCGGGCACC


 


2221
ACCTCCTGAG AGCCCATGCC TTGGCTTGGC ATCTGTACGA TGACAAGTTT AGGGCGGCTC


 


2281
AGAAAGGCAA AATATCCATC GCCTTGCAGG CTGACTGGAT AGAACCGGCC TGCCCTTTCT


 


2341
CTCAAAATGA CAAAGAAGTG GCCGAGAGAG TTTTGGAATT TGATATAGGC TGGCTGGCAG


 


2401
AGCCTATTTT TGGTTCCGGA GATTATCCAC GTGTGATGAG GGACTGGCTG AACCAAAAAA


 


2461
ACAATTTTCT TTTGCCCTAT TTCACCGAAG ATGAAAAAAA GCTAGTCCGG GGTTCCTTTG


 


2521
ACTTCCTGGC GGTGAGTCAT TACACCACCA TTCTGGTAGA CTGGGAAAAG GAGGATCCGA


 


2581
TGAAATACAA CGATTACTTG GAGGTACAGG AGATGACTGA CATCACATGG CTCAACTCTC


 


2641
CCAGTCAGGT GGCAGTGGTG CCTTGGGGGC TGCGCAAAGT GCTCAACTGG CTAAGGTTCA


 


2701
AGTACGGAGA CCTCCCGATG TATGTGACAG CCAATGGAAT CGATGATGAC CCCCACGCCG


 


2761
AGCAAGACTC ACTGAGGATC TATTATATTA AGAATTATGT GAATGAGGCT CTGAAAGCCT


 


2821
ACGTGTTGGA CGACATCAAC CTTTGTGGCT ACTTTGCGTA TTCACTTAGT GATCGCTCAG


 


2881
CTCCCAAGTC TGGCTTTTAT CGATATGCTG CGAATCAGTT TGAGCCCAAA CCATCTATGA


 


2941
AACATTACAG GAAAATTATT GACAGCAATG GCTTCCTGGG TTCTGGAACA CTGGGAAGGT


 


3001
TTTGTCCAGA AGAATACACT GTGTGCACCG AATGTGGATT TTTTCAAACC CGGAAGTCTT


 


3061
TGCTGGTCTT CATCTCGTTT CTTGTTTTTA CTTTTATTAT TTCTCTTGCT CTCATTTTTC


 


3121
ACTACTCCAA GAAAGGCCAG AGAAGTTATA AGTAA






In one embodiment, the FGFR is FGFR1c, FGFR3c, or FGFR4. In one embodiment of the present invention, the FGF receptor is FGFR1c receptor. In one particular embodiment, the FGFR1c receptor is the human FGFR1c receptor (GenBank Accession No. NP_075598, which is hereby incorporated by reference in its entirety). In another embodiment, the FGF receptor is FGFR3c receptor. In one particular embodiment, the FGFR3c receptor is the human FGFR3c receptor (GenBank Accession No. NP_000133, which is hereby incorporated by reference in its entirety). In another embodiment, the FGF receptor is FGFR4 receptor. In one particular embodiment, the FGFR4 receptor is the human FGFR4 receptor (GenBank Accession No. NP_002002, which is hereby incorporated by reference in its entirety).
In one embodiment, the method of facilitating FGFR-αKlotho co-receptor complex formation is carried out in vitro. In one embodiment, the method is carried out in a cell ectopically expressing αKlotho co-receptor and one or more of the cognate FGFRs of FGF23, which are FGFR1c, FGFR3c, and FGFR4. In one particular embodiment, the interleukin-3-dependent murine pro-B BaF3 cell line is used for ectopic expression of αKlotho co-receptor and one or more of the cognate FGFRs of FGF23. In one embodiment, the method is carried out in a cell endogenously expressing αKlotho co-receptor and one or more of the cognate FGFRs of FGF23. In one embodiment, the method is carried out in a renal cell, a parathyroid cell, a blood cell, a thymus cell, a pituitary cell, a hypothalamus-derived cell, a cell derived from the corpus striatum, and/or a cell derived from the cerebrum. In one particular embodiment, the method is carried out in a renal proximal tubule epithelial cell.
In one embodiment, the method of facilitating FGFR-αKlotho co-receptor complex formation is carried out in vivo. In one embodiment, the method is carried out in a mammal. In one particular embodiment, the mammal is a mouse. In one embodiment, the mouse is an fgf23-gene knockout mouse. In one embodiment, serum concentration of phosphate is used as readout for the method. In one embodiment, renal excretion of phosphate is used as readout for the method. In one embodiment, serum concentration of bioactive vitamin D is used as readout for the method. In one embodiment, renal expression of 1α-hydroxylase (CYP27B1) is used as readout for the method. In one embodiment, renal expression of NaPi-2A and/or NaPi-2C is used as readout for the method.
Yet a further aspect of the present invention relates to a method of screening for agents capable of facilitating fibroblast growth factor receptor (“FGFR”)-αKlotho co-receptor complex formation in the treatment of a disorder. This method involves providing a chimeric FGF that includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine FGF and the C-terminus includes a C-terminal portion of FGF23. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. This method also involves providing a binary αKlotho-FGFR complex and providing one or more candidate agents. This method further involves combining the chimeric FGF, the binary αKlotho-FGFR complex, and the one or more candidate agents under conditions permitting the formation of a ternary complex between the chimeric FGF and the binary αKlotho-FGFR complex in the absence of the one or more candidate agents. This method also involves identifying the one or more candidate agents that decrease ternary complex formation between the chimeric FGF and the binary αKlotho-FGFR compared to the ternary complex formation in the absence of the one or more candidate agents as suitable for treating the disorder.
In one embodiment the FGF molecule is FGF1 or FGF2. In one embodiment, the FGF protein is an FGF protein that possesses intrinsically greater binding affinity for FGF receptor than a native endocrine FGF ligand. In one embodiment, the FGF protein is an FGF protein that possesses intrinsically greater thermal stability than a native endocrine FGF ligand. In one embodiment, the method involves introducing one or more modifications to the FGF protein, where the modification alters receptor-binding specificity and/or receptor-binding affinity of the FGF protein. In one embodiment, the method involves introducing one or more modifications to the FGF protein, where the modification alters the stability of the FGF protein. For example, receptor-binding specificity of FGF1, which by nature binds to all the seven principal FGFRs, may be altered to, for example, reduce any risk for adverse effects (e.g., mitogenicity). Paracrine FGFs, portions of paracrine FGFs, and modifications thereto are described above.
Suitable chimeric proteins for use in accordance with this aspect of the present invention are described above and throughout the present application. Suitable paracrine FGFs, as well as suitable modifications to decrease binding affinity for heparin and/or heparan sulfate, to alter receptor-binding specificity and/or to alter receptor-binding affinity compared to the portion without the modification, are also described above.
In one embodiment, the modulation is a competitive interaction between the chimeric FGF molecule and the one or more candidate agents for binding to the binary αKlotho-FGFR complex.
In one embodiment, the FGFR is FGFR1c, FGFR3c, or FGFR4.
In one embodiment, the disorder is associated with hyperphosphatemia, abnormally high renal phosphate reabsorption, abnormally low blood levels of full-length, bioactive FGF23, inappropriately normal blood levels of bioactive vitamin D, and/or elevated blood levels of bioactive vitamin D. In one embodiment, the disorder is associated with soft tissue calcification.
In one embodiment of the screening aspects of the present invention, a plurality of compounds or agents is tested. Candidate agents may include small molecule compounds or larger molecules (e.g., proteins or fragments thereof). In one embodiment, the candidate compounds are biomolecules. In one embodiment, the biomolecules are proteins. In one embodiment, the biomolecules are peptides. In one embodiment, the candidates are peptides or peptide mimetics having similar structural features to native FGF ligand. In one embodiment, the candidate agent is a second chimeric FGF molecule. In one particular embodiment, the peptides are synthetic peptides. In one embodiment, the compounds are small organic molecules.
In one embodiment of the screening aspects of the present invention, the method is carried out using a cell-based assay. In one embodiment, the identifying is carried out using a cell-based assay.
In one embodiment of the screening aspects of the present invention, the method is carried out using a binding assay. In one embodiment, the binding assay is a direct binding assay. In one embodiment, the binding assay is a competition-binding assay. In one embodiment, the modulation stabilizes the ternary complex between the chimeric FGF molecule and the binary αKlotho-FGFR complex. In one embodiment, the stabilization is compared to the native ternary complex.
In one embodiment, the modulation is an allosteric or kinetic modulation. In one embodiment, the allosteric or kinetic modulation is compared to the native ternary complex. Such stabilization or allosteric or kinetic modulation can be measured modulation determined according to methods known in the art (e.g., by use of surface plasmon resonance (SPR) spectroscopy experiments as described in the Examples infra).
In one embodiment, the binding assay is carried out using surface plasmon resonance spectroscopy. In one embodiment, the identifying is carried out using a binding assay. In one embodiment, the identifying is carried out using surface plasmon resonance spectroscopy.
In one embodiment of the screening aspects of the present invention, the cell-based assay is carried out with renal cells. In one particular embodiment, the renal cells are proximal tubule epithelial cells. In one embodiment, the cell-based assay is carried out with parathyroid cells. In one embodiment, the cell-based assay is carried out with blood cells. In one embodiment, the cell-based assay is carried out with thymus cells. In one embodiment, the cell-based assay is carried out with pituitary cells. In one embodiment, the cell-based assay is carried out with cells derived from the hypothalamus. In one embodiment, the cell-based assay is carried out with cells derived from the corpus striatum. In one embodiment, the cell-based assay is carried out with cells derived from the cerebrum. In one embodiment, inhibition of phosphate uptake by the cells is the assay readout. In one embodiment, repression of the NaPi-2A gene and/or the NaPi-2C gene is the assay readout. In one embodiment, reduction of NaPi-2A and/or NaPi-2C protein expression is the assay readout. In one embodiment, reduction of NaPi-2A and/or NaPi-2C protein in the cell membrane is the assay readout. In one embodiment, repression of the CYP27B1 gene is the assay readout. In one embodiment, a dose-response curve is generated for inhibition of phosphate uptake (repression of the NaPi-2A gene and/or the NaPi-2C gene, reduction of NaPi-2A and/or NaPi-2C protein expression, reduction of NaPi-2A and/or NaPi-2C protein in the cell membrane, repression of the CYP27B1 gene) by a candidate compound to determine potency and efficacy of the candidate compound. For example, if the dose-response curve is shifted to the left compared to that obtained for the chimeric FGF protein, the candidate compound is more potent than the chimeric FGF protein and/or native FGF23. In one embodiment, an IC50 value is derived from the dose-response curve of a candidate compound to determine potency of the candidate compound. An IC50 value smaller than that obtained for the chimeric FGF protein identifies a candidate compound as more potent than the chimeric FGF protein and/or native FGF23.
In one embodiment of the screening aspects of the present invention, the cell-based assay is carried out with mammalian cells ectopically expressing αKlotho. In one particular embodiment, the cells are HEK293 cells. In one embodiment, activation of FGF receptor is the assay readout. In one embodiment, tyrosine phosphorylation of an FGF receptor substrate is used as readout for FGF receptor activation. In one particular embodiment, the FGF receptor substrate is FGF receptor substrate 2α. In one embodiment, activation of downstream mediators of FGF signaling is used as readout for (or an indicator of) FGF receptor activation. In one particular embodiment, the downstream mediator of FGF signaling is 44/42 mitogen-activated protein kinase. In one embodiment, the downstream mediator of FGF signaling is a transcription factor. In one particular embodiment, the transcription factor is early growth response 1. In one embodiment, a dose-response curve is generated for αKlotho-dependent activation of FGF receptor by a candidate compound to determine potency and efficacy of the candidate compound. For example, if the dose-response curve is shifted to the left compared to that obtained for the chimeric FGF protein, the candidate compound is more potent than the chimeric FGF protein and/or native FGF23. In one embodiment, an IC50 value is derived from the dose-response curve of a candidate compound to determine potency of the candidate compound. An IC50 value smaller than that obtained for the chimeric FGF protein identifies a candidate compound as more potent than the chimeric FGF protein and/or native FGF23.
In one embodiment of the screening aspects of the present invention, the surface plasmon resonance spectroscopy-based assay is carried out using the chimeric FGF protein as ligand coupled to a biosensor chip. In one embodiment, mixtures of the binary complex of FGFR ligand-binding domain and αKlotho ectodomain with increasing concentrations of a candidate compound are passed over a biosensor chip containing chimeric FGF protein. In one particular embodiment, the FGFR ligand-binding domain is the FGFR1c ligand-binding domain. In one embodiment, an inhibition-binding curve is plotted for a candidate compound to determine potency of the candidate compound. For example, if the inhibition-binding curve is shifted to the left compared to that obtained for the chimeric FGF protein, the candidate compound has greater potency than the chimeric FGF protein and/or native FGF23. In one embodiment, an IC50 value is derived from the inhibition-binding curve of a candidate compound to determine potency of the candidate compound. An IC50 value smaller than that obtained for containing chimeric FGF protein identifies a candidate compound as more potent than the chimeric FGF protein and/or native FGF23. In one embodiment, the inhibition constant Ki is determined for a candidate compound to determine potency of the candidate compound. A Ki value smaller than that obtained for native FGF23 identifies a candidate compound as more potent than the chimeric FGF protein and/or native FGF23.
Yet another aspect of the present invention relates to a modified FGF23 protein. The modified FGF23 protein includes an FGF23 protein that includes a modification to decrease binding affinity for heparin and/or heparan sulfate compared to an FGF23 protein without the modification.
FGF23 proteins suitable for use in accordance with this aspect of the present invention include those described above (i.e., human FGF23 and orthologs thereof). In one embodiment, the modified FGF23 is derived from a mammalian FGF23. In one embodiment, the modified FGF23 protein includes an FGF protein that includes the amino acid sequence of SEQ ID NO: 233, where the modification includes a substitution at amino acid residues selected from R48, N49, R140, R143, and combinations thereof. In one embodiment, the modification includes one or more substitutions selected from R48A/G/S, N49A/G/S, R140A/G/S, R143A/G/S, and combinations thereof. In one embodiment, the modified FGF23 protein has an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity or homology to the amino acid sequence of SEQ ID NO: 233. In one embodiment, the modified FGF23 protein has an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity or homology to an ortholog of human FGF23 (SEQ ID NO:233). In one embodiment, the modified FGF23 includes a modification to decrease binding affinity for heparin and/or heparan sulfate compared to an FGF23 protein without the modification and retains biological activity of native FGF23. In one embodiment, the modified FGF23 includes a modification to decrease binding affinity for heparin and/or heparan sulfate compared to an FGF23 protein without the modification and retains the ability to bind a binary αKlotho-FGFR complex.
In one embodiment, the modification includes a substitution at amino acid residues corresponding to positions of SEQ ID NO: 233 selected from R48, N49, R140, R143, and combinations thereof. In one embodiment, the modification includes one or more substitutions selected from R48A/G/S, N49A/G/S, R140A/G/S, R143A/G/S, and combinations thereof.
Another aspect of the present invention relates to a pharmaceutical composition including the modified FGF23 protein according to the present invention and a pharmaceutically-acceptable carrier. Suitable pharmaceutical compositions, dosages, carriers and the like are described above.
In one embodiment, the pharmaceutical composition further includes a hypophosphatemic agent, a phosphate binder, a vitamin D antagonist, an analgesic, and/or an anti-inflammatory agent.
In one embodiment, the pharmaceutical composition further includes an organotropic targeting agent. In one embodiment, the targeting agent is covalently linked to the chimeric protein via a linker that is cleaved under physiological conditions.
As noted above, chimeric and/or modified proteins according to the present invention may also be modified using one or more additional or alternative strategies for prolonging the in vivo half-life of the protein. One such strategy involves the generation of D-peptide chimeric proteins, which consist of unnatural amino acids that are not cleaved by endogenous proteases. Alternatively, the chimeric and/or modified proteins may be fused to a protein partner that confers a longer half-life to the protein upon in vivo administration. Suitable fusion partners include, without limitation, immunoglobulins (e.g., the Fc portion of an IgG), human serum albumin (HAS) (linked directly or by addition of the albumin binding domain of streptococcal protein G), fetuin, or a fragment of any of these. The chimeric and/or modified proteins may also be fused to a macromolecule other than protein that confers a longer half-life to the protein upon in vivo administration. Suitable macromolecules include, without limitation, polyethylene glycols (PEGs).
In one embodiment, the modified FGF23 is fused at its N-terminus to an agent that increases the half-life of the modified FGF23 protein in circulation. In one embodiment, the agent that increases the half-life is a PEG molecule. In one embodiment, the agent that increases the half-life is an antibody fragment.
Another aspect of the present invention relates to a method for treating a subject suffering from a disorder. This method involves selecting a subject suffering from the disorder and administering to the selected subject a therapeutically effective amount of a modified FGF23 protein including a modification to decrease binding affinity for heparin and/or heparan sulfate compared to an FGF23 protein without the modification.
In one embodiment, the disorder is associated with hyperphosphatemia, abnormally high renal phosphate reabsorption, abnormally low blood levels of full-length, bioactive FGF23, inappropriately normal blood levels of bioactive vitamin D, and/or elevated blood levels of bioactive vitamin D. In one embodiment, the disorder is associated with soft tissue calcification. Such disorders and methods for evaluating those disorders are described above and will be known to those of skill in the art. Suitable modes of administration are also described above.
In one embodiment, the modified FGF23 protein is administered with a pharmaceutically-acceptable carrier.
In one embodiment, the selected subject is a mammal. In one embodiment, the selected subject is a human.
In one embodiment, the modified FGF23 protein is co-administered with a hypophosphatemic agent, a phosphate binder, a vitamin D antagonist, an analgesic, and/or an anti-inflammatory agent.
EXAMPLES
Example 1
Purification of FGF, FGFR, and Klotho Proteins
The N-terminally hexahistidine-tagged, mature form of human FGF19 (SEQ ID NO: 333) (R23 to K216), human FGF21 (SEQ ID NO: 334) (H29 to S209; FIG. 5A), and human FGF23 (Y25 to I251; FIG. 5A) was refolded in vitro from bacterial inclusion bodies, and purified by published protocols (Ibrahimi et al., Hum. Mol. Genet. 13:2313-2324 (2004); Plotnikov et al., Cell 101:413-424 (2000), which is hereby incorporated by reference in its entirety). The amino acid sequence of human FGF19 (SEQ ID NO:333) (GenBank Accession No. NP_005108, which is hereby incorporated by reference in its entirety) is as follows:









1
MRSGCVVVHV WILAGLWLAV AGRPLAFSDA GPHVHYGWGD PIRLRHLYTS GPHGLSSCFL



 


61
RIRADGVVDC ARGQSAHSLL EIKAVALRTV AIKGVHSVRY LCMGADGKMQ GLLQYSEEDC


 


121
AFEEEIRPDG YNVYRSEKHR LPVSLSSAKQ RQLYKNRGFL PLSHFLPMLP MVPEEPEDLR


 


181
GHLESDMFSS PLETDSMDPF GLVTGLEAVR SPSFEK






The amino acid sequence of human FGF21 (SEQ ID NO: 334) (GenBank Accession No. NP_061986, which is hereby incorporated by reference in its entirety), as follows:









1
MDSDETGFEH SGLWVSVLAG LLLGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH



 


61
LEIREDGTVG GAADQSPESL LQLKALKPGV IQILGVKTSR FLCQRPDGAL YGSLHFDPEA


 


121
CSFRELLLED GYNVYQSEAH GLPLHLPGNK SPHRDPAPRG PARFLPLPGL PPALPEPPGI


 


181
LAPQPPDVGS SDPLSMVGPS QGRSPSYAS.






FIG. 11 shows an alignment of the C-terminal tail sequences of human FGF19, FGF21, and FGF23.
HS-binding site mutants of FGF19 (K149A) and FGF23 (R140A/R143A) were purified from bacterial inclusion bodies by similar protocols as the wild-type proteins. In order to minimize proteolysis of FGF23 wild-type and mutant proteins, arginine residues 176 and 179 of the proteolytic cleavage site 176RXXR179 were replaced with glutamine as it occurs in the phosphate wasting disorder “autosomal dominant hypophosphatemic rickets” (ADHR) (White et al., Nat. Genet. 26:345-348 (2000); White et al., Kidney Int. 60:2079-2086 (2001), which are hereby incorporated by reference in their entirety). Human FGF1 (SEQ ID NO: 1) (M1 to D155; FIG. 6), N-terminally truncated human FGF1 (K25 to D155, termed FGF1ΔNT; FIG. 6), human FGF2 (SEQ ID NO: 121) (M1 to S155; FIG. 5A), and human FGF homologous factor 1B (FHF1B; M1 to T181) were purified by published protocols (Plotnikov et al., Cell 101:413-424 (2000); Olsen et al., J. Biol. Chem. 278:34226-34236 (2003), which are hereby incorporated by reference in their entirety).
Chimeras composed of the core domain of FGF2 (M1 to M151) and the C-terminal region of either FGF21 (P168 to S209) or FGF23 (R161 to I251) (termed FGF2WTcore-FGF21C-tail and FGF2WTcore-FGF23C-tail, respectively; FIG. 5A) were purified by the same protocol as that for native FGF2 (Plotnikov et al., Cell 101:413-424 (2000), which is hereby incorporated by reference in its entirety). Analogous chimeras containing three mutations in the HS-binding site of the FGF2 core (K128D/R129Q/K134V) (termed FGF2ΔHBScore-FGF21C-tail and FGF2ΔHBScore-FGF23C-tail respectively, FIG. 5A) were purified from the soluble bacterial cell lysate fraction by ion-exchange and size-exclusion chromatographies. In order to minimize proteolysis of the chimeras containing the C-terminal sequence from R161 to I251 of FGF23, arginine residues 176 and 179 of the proteolytic cleavage site 176RXXR179 located within this sequence were replaced with glutamine as it occurs in ADHR (White et al., Nat. Genet. 26:345-348 (2000); White et al., Kidney Int. 60:2079-2086 (2001), which are hereby incorporated by reference in their entirety). In addition, in order to prevent disulfide-mediated dimerization of FGF2 and chimeric FGF2 proteins, cysteine residues 78 and 96 were mutated to serine. An HS-binding site mutant of FGF1 (K127D/K128Q/K133V) (termed FGF1ΔHBScore; FIG. 6) and chimeras composed of the core domain of the HS-binding site mutant of FGF1 (M1 to L150, K127D/K128Q/K133V) and the C-terminal region of either FGF19 (L169 to K216) or FGF21 (P168 to S209) (termed FGF1ΔHBScore-FGF19C-tail and FGF1ΔHBScore-FGF21C-tail, respectively; FIG. 6) were purified from the soluble bacterial cell lysate fraction by ion-exchange and size-exclusion chromatographies. The N-terminally hexahistidine-tagged C-terminal tail peptide of FGF23 (S180 to I251, termed FGF23C-tail) was purified by a published protocol (Goetz et al., Proc. Nat'l. Acad. Sci. U.S.A. 107:407-412 (2010), which is hereby incorporated by reference in its entirety). The ligand-binding domain of human FGFR1c (D142 to R365) was refolded in vitro from bacterial inclusion bodies, and purified by published protocols (Ibrahimi et al., Hum. Mol. Genet. 13:2313-2324 (2004); Plotnikov et al., Cell 101:413-424 (2000), which are hereby incorporated by reference in their entirety). The ectodomain of murine αKlotho (A35 to K982) and the ectodomain of murine βKlotho (F53 to L995) were expressed in HEK293 cells as fusion proteins with a C-terminal FLAG tag (Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Kurosu et al., Science 309:1829-1833 (2005), which are hereby incorporated by reference in their entirety). The binary complex of FGFR1c ligand-binding domain with αKlotho ectodomain (referred to as αKlotho-FGFR1c complex) was prepared by a published protocol (Goetz et al., Proc. Nat'l. Acad. Sci. U.S.A. 107:407-412 (2010), which is hereby incorporated by reference in its entirety). The binary complex of FGFR1c ligand-binding domain with βKlotho ectodomain (referred to as βKlotho-FGFR1c complex) was prepared in the same fashion as the αKlotho-FGFR1c complex.
Example 2
Analysis of FGF-Heparin and FGF-FGFR-α/βKlotho Interactions by Surface Plasmon Resonance Spectroscopy
Surface plasmon resonance (SPR) experiments were performed on a Biacore 2000 instrument (Biacore AB), and the interactions were studied at 25° C. in HBS-EP buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) polysorbate 20). To study endocrine FGF-heparin interactions, a heparin chip was prepared by immobilizing biotinylated heparin (Sigma-Aldrich) on flow channels of a research-grade streptavidin chip (Biacore AB). The coupling density was  ̃5 fmol mm−2 of flow channel. To measure binding of chimeric FGF2 proteins to heparin, biotinylated heparin was coupled to a streptavidin chip at an approximately 4-fold lower density as judged based on the binding responses obtained for FGF1. To study FGF-FGFR-α/βKlotho interactions, FGF chips were prepared by covalent coupling of FGF proteins through their free amino groups on flow channels of research grade CM5 chips (Biacore AB). Proteins were injected over a chip at a flow rate of 50 μl min−1, and at the end of each protein injection (180 and 300 s, respectively), HBS-EP buffer (50 μl min−1) was flowed over the chip to monitor dissociation for 180 or 240 s. The heparin chip surface was regenerated by injecting 50 μl of 2.0 M NaCl in 10 mM sodium acetate, pH 4.5. For FGF chips, regeneration was achieved by injecting 2.0 M NaCl in 10 mM sodium/potassium phosphate, pH 6.5. To control for nonspecific binding in experiments where an FGF ligand was immobilized on the chip, FHF1B, which shares structural similarity with FGFs but does not exhibit any FGFR binding (Olsen et al., J. Biol. Chem. 278:34226-34236 (2003), which is hereby incorporated by reference in its entirety), was coupled to the control flow channel of the chip ( ̃15-30 fmol mm−2). In experiments where heparin was immobilized on the chip, the control flow channel was left blank. The data were processed with BiaEvaluation software (Biacore AB). For each protein injection over the heparin chip, the nonspecific responses from the control flow channel were subtracted from the responses recorded for the heparin flow channel. Similarly, for each protein injection over a FGF chip, the nonspecific responses from the FHF1B control flow channel were subtracted from the responses recorded for the FGF flow channel. Where possible, equilibrium dissociation constants (KDs) were calculated from fitted saturation binding curves. Fitted binding curves were judged to be accurate based on the distribution of the residuals (even and near zero) and χ2 (<10% of Rmax).
To examine whether the K149A mutation abrogates residual heparin binding of FGF19, increasing concentrations of wild-type FGF19 were passed over a heparin chip. Thereafter, the FGF19K149A mutant was injected over the heparin chip at the highest concentration tested for the wild-type ligand. The effect of the R140A/R143A double mutation in the HS-binding site of FGF23 on residual heparin binding of FGF23 was examined in the same fashion as was the effect of the HS-binding site mutation in FGF19.
To verify that the K128D/R129Q/K134V triple mutation in the HS-binding site of the FGF2 core domain diminishes heparin-binding affinity of the FGF2 core, increasing concentrations of FGF2ΔHBScore-FGF21C-tail and FGF2ΔHBScore-FGF23C-tail were passed over a heparin chip. As a control, binding of FGF2WTcore-FGF21C-tail and FGF2WTcore-FGF23C-tail to heparin was studied.
To examine whether the FGF2ΔHBScore-FGF23C-tail chimera can compete with FGF23 for binding to the αKlotho-FGFR1c complex, FGF23 was immobilized on a chip ( ̃16 fmol mm−2 of flow channel). Increasing concentrations of FGF2ΔHBScore-FGF23C-tail were mixed with a fixed concentration of αKlotho-FGFR1c complex in HBS-EP buffer, and the mixtures were injected over the FGF23 chip. As controls, the binding competition was carried out with FGF23 or FGF2 as the competitor in solution. As an additional specificity control, competition of the FGF2ΔHBScore-FGF23C-tail chimera with FGF21 for binding to the αKlotho-FGFR1c complex was studied. αKlotho-FGFR1c complex was mixed with FGF2ΔHBScore-FGF23C-tail or FGF23 at a molar ratio of 1:10, and the mixture was injected over a chip containing immobilized FGF21 ( ̃12 fmol mm−2 of flow channel).
To test whether the FGF2ΔHBScore-FGF21C-tail chimera can compete with FGF21 for binding to the βKlotho-FGFR1c complex, increasing concentrations of FGF2ΔHBScore-FGF21C-tail were mixed with a fixed concentration of βKlotho-FGFR1c complex in HBS-EP buffer, and the mixtures were passed over a chip containing immobilized FGF21 ( ̃19 fmol mm−2 of flow channel). As controls, the binding competition was carried out with FGF21 or FGF2 as the competitor in solution. As an additional specificity control, competition of the FGF2ΔHBScore-FGF21C-tail chimera with FGF23 for binding to the αKlotho-FGFR1c complex was studied. αKlotho-FGFR1c complex was mixed with FGF2ΔHBScore-FGF21C-tail or FGF21 at a molar ratio of 1:10, and the mixture was injected over a chip containing immobilized FGF23 ( ̃12 fmol mm−2 of flow channel).
To measure binding of FGFR1c to each of the three endocrine FGFs, increasing concentrations of FGFR1c ligand-binding domain were injected over a chip containing immobilized FGF19, FGF21, and FGF23 ( ̃30 fmol mm−2 of flow channel). As a control, binding of FGFR1c to FGF2 immobilized on a chip was studied. As additional controls, binding of the αKlotho-FGFR1c complex to FGF23 and binding of FGFR1c to the C-terminal tail peptide of FGF23 was measured.
Example 3
Analysis of Phosphorylation of FRS2α and 44/42 MAP Kinase in Hepatoma and Epithelial Cell Lines
To examine whether the FGF19K149A and FGF23R140A/R143A mutants can activate FGFR in a α/βKlotho-dependent fashion, induction of tyrosine phosphorylation of FGFR substrate 2α (FRS2α) and downstream activation of MAP kinase cascade was used as readout for FGFR activation. Subconfluent cells of the H4IIE rat hepatoma cell line, which endogenously expresses βKlotho (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007), which is hereby incorporated by reference in its entirety), were serum starved for 16 h and then stimulated for 10 min with the FGF19K149A mutant or wild-type FGF19 (0.2 ng ml−1 to 2.0 μg ml−1). Similarly, subconfluent cells of a HEK293 cell line ectopically expressing the transmembrane isoform of murine αKlotho (Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006), which is hereby incorporated by reference in its entirety) were treated with the FGF23R140A/R143A mutant or wild-type FGF23 (0.1 to 100 ng ml−1). After stimulation, the cells were lysed (Kurosu et al., Science 309:1829-1833 (2005), which is hereby incorporated by reference in its entirety), and cellular proteins were resolved on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The protein blots were probed with antibodies to phosphorylated FRS2α, phosphorylated 44/42 MAP kinase, total (phosphorylated and nonphosphorylated) 44/42 MAP kinase, and αKlotho. Except for the anti-αKlotho antibody (KM2119) (Kato et al., Biochem. Biophys. Res. Commun. 267:597-602 (2000), which is hereby incorporated by reference in its entirety), all antibodies were from Cell Signaling Technology.
Example 4
Analysis of Egr1 Protein Expression in an Epithelial Cell Line
To examine whether the FGF2ΔHBScore-FGF21C-tail and FGF2ΔHBScore-FGF23C-tail chimeras can activate FGFR in a HS-dependent fashion, induction of protein expression of the transcription factor early growth response 1 (Egr1), a known downstream mediator of FGF signaling, was used as readout for FGFR activation. HEK293 cells were serum starved overnight and then stimulated for 90 min with FGF2ΔHBScore-FGF21C-tail or FGF2ΔHBScore-FGF23C-tail (0.1 and 0.3 nM). Cell stimulation with FGF2WTcore-FGF21C-tail, FGF2WTcore-FGF23C-tail, FGF21, and FGF23 served as controls. To test whether the FGF2ΔHBScore-FGF21C-tail chimera can activate FGFR in a βKlotho-dependent fashion, HEK293 cells transfected with murine βKlotho were serum starved overnight and then stimulated for 90 min with FGF2ΔHBScore-FGF21C-tail or FGF21 (3 to 300 ng ml−1). After stimulation, the cells were lysed (Kurosu et al., Science 309:1829-1833 (2005), which is hereby incorporated by reference in its entirety), and cellular proteins were resolved on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The protein blots were probed with antibodies to Egr1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The anti-Egr1 antibody was from Cell Signaling Technology and the anti-GAPDH antibody was from Abcam.
Example 5
Analysis of CYP7A1 and CYP8B1 mRNA Expression in Murine Liver Tissue
To examine the metabolic activity of the FGF19K149A mutant in vivo, 6- to 8-week old C57BL/6 mice were fasted overnight and then given intraperitoneally a single dose (1 mg kg body weight−1) of FGF19K149A or FGF19 as a control. 6 h after the injection, the mice were sacrificed, and liver tissue was excised and frozen. Total RNA was isolated from liver tissue, and mRNA levels of cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) were measured using quantitative real time RT-PCR as described previously (Inagaki et al., Cell Metab. 2:217-225 (2005); Kim et al., J. Lipid Res. 48:2664-2672 (2007), which are hereby incorporated by reference in their entirety). The Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center at Dallas had approved the experiments.
Example 6
Measurement of Serum Phosphate in Mice
The metabolic activity of the FGF23R140A/R143A mutant was examined both in normal mice and in Fgf23 knockout mice. 4- to 5-week old C57BL/6 mice were given intraperitoneally a single dose (0.29 mg kg body weight−1) of FGF23R140A/R143A or FGF23 as a control. Before the injection and 8 h after the injection, blood was drawn from the cheek pouch and spun at 3,000×g for 10 min to obtain serum. Phosphate concentration in serum was measured using the Phosphorus Liqui-UV Test (Stanbio Laboratory). 6- to 8-week old Fgf23 knockout mice (Sitara et al., Matrix Biol. 23:421-432 (2004), which is hereby incorporated by reference in its entirety) (56) were given two injections of FGF23R140A/R143A or FGF23 at 8 h intervals (0.71 mg kg body weight−1 each), and blood samples were collected for phosphate analysis before the first injection and 8 h after the second injection.
To test whether the FGF2ΔHBScore-FGF23C-tail chimera exhibits FGF23-like metabolic activity, 5- to 6-week old C57BL/6 mice were given a single injection of FGF2ΔHBScore-FGF23C-tail (0.21 mg kg body weight−1). As controls, mice were injected with FGF2WTcore-FGF23C-tail or FGF23. Before the injection and 8 h after the injection, blood samples were collected for measurement of serum phosphate. To confirm that αKlotho is required for the metabolic activity of the FGF2ΔHBScore-FGF23C-tail chimera, 7- to 8-week old αKlotho knockout mice (Lexicon Genetics) were injected once with FGF2ΔHBScore-FGF23C-tail or FGF23 as a control (0.51 mg kg body weight−1). Before the injection and 8 h after the injection, blood samples were collected for phosphate analysis. The Harvard University Animal Care and Research committee board had approved all the experiments.
Example 7
Analysis of CYP27B1 mRNA Expression in Murine Renal Tissue
The ability of the FGF2ΔHBScore-FGF23C-tail chimera to reduce renal expression of 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1) was used as another readout for FGF23-like metabolic activity. C57BL/6 mice injected with FGF2ΔHBScore-FGF23C-tail, FGF2WTcore-FGF23C-tail, or FGF23 were sacrificed 8 h after the protein injection, and renal tissue was excised and frozen. CYP27B1 mRNA levels in total renal tissue RNA were measured using real time quantitative PCR as described previously (Nakatani et al., FASEB J. 23:3702-3711 (2009); Ohnishi et al., Kidney Int. 75:1166-1172 (2009), which are hereby incorporated by reference in their entirety). The Harvard University Animal Care and Research committee board had approved the experiments.
Example 8
Insulin Tolerance Test in Mice
The ability of the FGF2ΔHBScore-FGF21C-tail chimera to potentiate the hypoglycemic effect of insulin was used as readout for FGF21-like metabolic activity (Ohnishi et al., FASEB J. 25:2031-2039 (2011), which is hereby incorporated by reference in its entirety). 8- to 12-week old C57BL/6 mice were kept on normal chow. On the day of the insulin tolerance test, mice were fasted for 4 h and then bled from the cheek pouch for measuring fasting blood glucose levels. Thereafter, mice were administered intraperitoneally insulin (0.5 units kg body weight−1) alone or insulin (0.5 units·kg body weight−1) plus FGF2ΔHBScore-FGF21C-tail chimera (0.3 mg kg body weight−1). As a control, mice were co-injected with insulin plus FGF21. At the indicated time points after the injection (FIG. 7G), blood was drawn from the tail vein. Glucose concentrations in the blood samples were determined using Bayer Contour® blood glucose test strips (Bayer Corp.). The Harvard University Animal Care and Research committee board had approved the experiments.
Example 9
Analysis of Blood Glucose in ob/ob Mice
ob/ob mice were injected subcutaneously with FGF1ΔNT, FGF1ΔHBS, or FGF1ΔHBScore-FGF21C-tail chimera. Injection of native FGF1 or native FGF21 served as controls. A single bolus of 0.5 mg of protein per kg of body weight was injected. This dose was chosen on the basis that maximal efficacy of the hypoglycemic effect of native FGF1 is seen at this dose. Before the protein injection and at the indicated time points after the injection (FIGS. 9A-9C), blood glucose concentrations were measured using an OneTouch Ultra glucometer (Lifescan). The Institutional Animal Care and Use Committee at the Salk Institute for Biological Sciences at La Jolla had approved the experiments.
Example 10
Statistical Analysis
Data are expressed as mean±SEM. A Student's t test or analysis of variance (ANOVA) was used as appropriate to make statistical comparisons. A value of P<0.05 was considered significant.
Example 11
HS is Dispensable for the Metabolic Activity of FGF19 and FGF23
In order to engineer endocrine FGFs devoid of HS binding, the FGF19 crystal structure (PDB ID: 2P23; (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety) was compared with that of FGF2 bound to a heparin hexasaccharide (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)). This analysis shows that solvent-exposed residues K149, Q150, Q152, and R157 of FGF19 lie at the corresponding HS-binding site of this ligand, and hence could account for the residual HS binding of FGF19 (FIGS. 1A, 1B, and 2). Likewise, comparative analysis of the FGF23 crystal structure (PDB ID: 2P39; (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety)) with that of heparin-bound FGF2 (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)) points to R48, N49, R140, and R143 as candidates mediating the residual HS binding of this ligand (FIGS. 1A, 1C, and 2). In agreement with the structural predictions, replacement of K149 alone in FGF19 with alanine and combined substitution of R140 and R143 in FGF23 for alanine were sufficient to abolish residual HS binding of these ligands (FIGS. 3B-3G).
To test the impact of knocking out residual HS binding of FGF19 on the signaling by this ligand, H4IIE hepatoma cells were stimulated with the FGF19K149A mutant or wild-type FGF19. H4IIE cells endogenously express FGFR4 and βKlotho (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007), which is hereby incorporated by reference in its entirety), the cognate receptor and co-receptor, respectively, for FGF19. The FGF19K149A mutant was as effective as wild-type FGF19 in inducing tyrosine phosphorylation of FRS2α and downstream activation of MAP kinase cascade (FIG. 4A). These data show that elimination of residual HS binding has no impact on the ability of FGF19 to signal in cultured cells. To test whether the same holds true for FGF23 signaling, HEK293 cells, which naturally express two of the three cognate receptors of FGF23, namely FGFR1c and FGFR3c (Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006), which is hereby incorporated by reference in its entirety) were transfected with the transmembrane isoform of αKlotho, the co-receptor of FGF23. These cells were treated with the FGF23R140A/R143A double mutant or wild-type FGF23. The FGF23R140A/R143A mutant had the same capacity as wild-type FGF23 in inducing phosphorylation of FRS2α and downstream activation of MAP kinase cascade (FIG. 4B). These data show that similar to FGF19, FGF23 does not need to bind HS in order to activate FGFR in cultured cells.
To substantiate the findings in cells, the metabolic activity of wild-type and mutated ligands in vivo were compared. Mice were injected with the FGF19K149A mutant or wild-type FGF19 and liver gene expression of CYP7A1 and CYP8B1, which are key enzymes in the major bile acid biosynthetic pathway (Russell, D. W., Annu. Rev. Biochem. 72:137-174 (2003), which is hereby incorporated by reference in its entirety), was analyzed. Like wild-type FGF19, the FGF19K149A mutant markedly decreased CYP7A1 and CYP8B1 mRNA levels (FIG. 4C), demonstrating that knockout of residual HS binding does not affect the metabolic activity of FGF19. To examine whether residual HS binding is also dispensable for the metabolic activity of FGF23, mice were injected with the FGF23R140A/R143A mutant or wild-type FGF23 and serum phosphate concentrations were measured. The FGF23R140A/R143A mutant reduced serum phosphate as effectively as wild-type FGF23 (FIG. 4D). Moreover, when injected into Fgf23 knockout mice, the FGF23R140A/R143A mutant exhibited as much of phosphate-lowering activity as wild-type FGF23 (FIG. 4D). These data show that, as in the case of FGF19, abolishment of residual HS binding does not impact the metabolic activity of FGF23 leading to the conclusion that HS is not a component of the endocrine FGF signal transduction unit (FIG. 1D).
Example 12
Conversion of a Paracrine FGF Into an Endocrine Ligand Confirms that HS is Dispensable for the Metabolic Activity of Endocrine FGFs
If HS is dispensable for the metabolic activity of endocrine FGFs, then it should be feasible to convert a paracrine FGF into an endocrine FGF by eliminating HS-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor binding site. Reducing HS-binding affinity will allow the ligand to freely diffuse and enter the blood circulation while attaching the C-terminal tail of an endocrine FGF will home the ligand into its target tissues. FGF2, a prototypical paracrine FGF, was chosen for conversion into FGF23-like and FGF21-like ligands, respectively. FGF2 was selected as paracrine ligand for this protein engineering exercise because it preferentially binds to the “c” isoform of FGFR1, the principal receptor mediating the metabolic activity of FGF23 (Gattineni et al., Am. J. Physiol. Renal Physiol. 297:F282-291 (2009); Liu et al., J. Am. Soc. Nephrol. 19:2342-2350 (2008), which are hereby incorporated by reference in their entirety) and FGF21 (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007), which is hereby incorporated by reference in its entirety), respectively. In the crystal structure of heparin-bound FGF2 (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)), K128, R129, and K134 mediate the majority of hydrogen bonds with heparin and hence mutation of these residues was predicted to cause a major reduction in HS-binding affinity of FGF2 (FIGS. 1A, 2, and 5A). Accordingly, these three residues were mutated and then the short C-terminal tail of the mutated FGF2 was replaced with the C-terminal tail of FGF23 (R161 to I251) or the C-terminal tail of FGF21 (P168 to S209) (FIG. 5A). The resulting chimeras were termed FGF2ΔHBScore-FGF2C-tail and FGF2ΔHBScore-FGF21C-tail (FIG. 5A). To demonstrate that reduction in HS-binding affinity is required for converting FGF2 into an endocrine ligand, two control chimeras were made in which the HS-binding site of the FGF2 core was left intact (FGF2WTcore-FGF23C-tail and FGF2WTcore-FGF21C-tail; FIG. 5A).
Consistent with the structural prediction, FGF2ΔHBScore-FGF23C-tail and FGF2ΔHBScore-FGF21C-tail exhibited poor binding affinity for HS compared to the corresponding control chimeras with intact HS-binding site (FIGS. 5B-5E). Since HS is an obligatory cofactor in paracrine FGF signaling, the FGF2ΔHBScore-FGF23C-tail and FGF2ΔHBScore-FGF21C-tail chimeras were predicted to lose the ability to activate FGFR1c in an HS-dependent fashion. To test this, HEK293 cells, which endogenously express FGFR1c, were stimulated with FGF2ΔHBScore-FGF23C-tail or FGF2WTcore-FGF23C-tail. Induction of protein expression of the transcription factor Egr1, a known downstream mediator of FGF signaling, was used as readout for FGFR activation. As shown in FIG. 5G, the FGF2ΔHBScore-FGF23C-tail chimera, like native FGF23, was ineffective in inducing Egr1 expression at concentrations at which the FGF2WTcore-FGF23C-tail chimera elicited a near maximal effect. The same observations were made for the FGF2ΔHBScore-FGF21C-tail chimera (FIG. 5F). These data show that, similar to native FGF23 and FGF21, the FGF2ΔHBScore-FGF23C-tail and FGF2ΔHBScore-FGF21C-tail chimeras lost the ability to activate FGFR in an HS-dependent, paracrine fashion.
To determine whether the FGF2ΔHBScore-FGF23C-tail and FGF2ΔHBScore-FGF21C-tail chimeras gained the ability to signal in a Klotho co-receptor-dependent, endocrine fashion, it was first analyzed whether these chimeras can form ternary complexes with FGFR1c and Klotho co-receptor. To this end, a SPR-based binding competition assay was employed. FGF23 was immobilized onto a SPR biosensor chip, and mixtures of a fixed concentration of binary αKlotho-FGFR1c complex with increasing concentrations of FGF2ΔHBScore-FGF23C-tail chimera were passed over the chip. FGF2ΔHBScore-FGF23C-tail competed, in a dose-dependent fashion, with immobilized FGF23 for binding to the αKlotho-FGFR1c complex (FIG. 7A), demonstrating that the chimera, like native FGF23 (FIG. 7B), is able to form a ternary complex with FGFR1c and αKlotho. To test whether the FGF2ΔHBScore-FGF21C-tail chimera can likewise form a ternary complex with FGFR1c and βKlotho, FGF21 was coupled to a SPR biosensor chip, and mixtures of the binary βKlotho-FGFR1c complex with FGF2ΔHBScore-FGF21C-tail were passed over the chip. FGF2ΔHBScore-FGF21C-tail effectively competed with immobilized FGF21 for binding to the βKlotho-FGFR1c complex (FIG. 8A), demonstrating that the chimera, like native FGF21 (FIG. 8B), is capable of binding to the binary complex of FGFR1c and βKlotho. Notably, native FGF2 failed to compete with FGF23 for binding to the αKlotho-FGFR1c complex (FIG. 7C), and with FGF21 for binding to the βKlotho-FGFR1c complex (FIG. 8C) since it lacks the Klotho co-receptor binding domain. To further confirm the binding specificity of the FGF2ΔHBScore-FGF2C-tail chimera for the αKlotho-FGFR1c complex, FGF2ΔHBScore-FGF2C-tail and βKlotho-FGFR1c complex were mixed at a molar ratio of 10:1, and the mixture was injected over a chip containing immobilized FGF21. FGF2ΔHBScore-FGF23C-tail, like native FGF23, failed to compete with FGF21 for binding to the βKlotho-FGFR1c complex (FIGS. 7D and 7E). Similarly, the FGF2ΔHBScore-FGF21C-tail chimera, like native FGF21, failed to compete with FGF23 for binding to the αKlotho-FGFR1c complex (FIGS. 8D and 8E). For the FGF2ΔHBScore-FGF21C-tail chimera, we investigated whether it is able to activate FGFR1c in a βKlotho-dependent fashion in cells. HEK293 cells were transfected with βKlotho and then stimulated with FGF2ΔHBScore-FGF21C-tail or FGF21. Similar to native FGF21, the FGF2ΔHBScore-FGF21C-tail chimera induced Egr1 protein expression in HEK293-βKlotho cells (FIG. 8F), indicating that the chimera is capable of activating FGFR1c in the presence of βKlotho.
To provide definite proof for the ligand conversion, the metabolic activity of the chimeras in vivo was tested. Specifically, the ability of the FGF2ΔHBScore-FGF23C-tail chimera to lower serum phosphate and to reduce renal gene expression of CYP27B1, which catalyzes the conversion of vitamin D into its bioactive form, was examined. Mice were injected with FGF2ΔHBScore-FGF23C-tail or as controls, FGF23 or FGF2WTcore-FGF23C-tail, and serum phosphate concentrations and renal CYP27B 1 mRNA levels were measured. Similar to native FGF23, the FGF2ΔHBScore-FGF23C-tail chimera caused a decrease in serum phosphate in wild-type mice (FIG. 7F). The chimera also induced a marked decrease in CYP27B1 mRNA levels, just like the native FGF23 ligand (FIG. 7G). These data show that the FGF2ΔHBScore-FGF23C-tail chimera acts as an FGF23-like hormone. Importantly, the FGF2WTcore-FGF23C-tail chimera failed to decrease serum phosphate or CYP27B1 mRNA levels (FIGS. 7F and 7G). This is expected because, owing to its high affinity for HS, this chimera should be trapped in the vicinity of the injection site and hence not be able to enter the blood circulation. Moreover, these data show that adding the Klotho co-receptor binding site is not sufficient to convert a paracrine FGF into an endocrine ligand. To confirm that the metabolic activity of the FGF2ΔHBScore-FGF23C-tail chimera is dependent on αKlotho, αKlotho knockout mice were injected with FGF2ΔHBScore-FGF23C-tail or FGF23 as a control, and serum concentrations of phosphate were measured. As shown in FIG. 7F, FGF2ΔHBScore-FGF23C-tail failed to lower serum phosphate, demonstrating that the chimera, like native FGF23 (FIG. 7F), requires αKlotho for metabolic activity.
To determine whether the FGF2ΔHBScore-FGF21C-tail chimera exhibits FGF21-like metabolic activity, its ability to potentiate the hypoglycemic effect of insulin was examined (Ohnishi et al., FASEB J. 25:2031-2039 (2011), which is hereby incorporated by reference in its entirety). Mice were injected with insulin plus FGF2ΔHBScore-FGF21C-tail, insulin plus FGF21, or insulin alone, and blood glucose concentrations were monitored for up to one hour after the injection. Similar to FGF21, the FGF2ΔHBScore-FGF21C-tail chimera enhanced the hypoglycemic effect of insulin (FIG. 8G), demonstrating that the chimera acts as an FGF21-like hormone.
To substantiate further the concept of FGF ligand conversion, another FGF21-like ligand was engineered using FGF1 as paracrine FGF, and the metabolic activity of the engineered protein was tested in vivo in a mouse model of diabetes and obesity. Besides serving as an additional proof-of-concept, the use of FGF1 for this particular ligand conversion was appealing because FGF1 on its own plays an essential role in glucose metabolism (Jonker et al., “A PPARγ-FGF1 Axis is Required for Adaptive Adipose Remodelling and Metabolic Homeostasis,” Nature 485:391-394 (2012), which is hereby incorporated by reference in its entirety). Notably, similar to FGF21, FGF1 is induced postprandially in gonadal white adipose tissue by the nuclear hormone receptor PPARγ (peroxisome proliferator activated receptor-γ) (Jonker et al., “A PPARγ-FGF1 Axis is Required for Adaptive Adipose Remodelling and Metabolic Homeostasis,” Nature 485:391-394 (2012); Dutchak et al., “Fibroblast Growth Factor-21 Regulates PPARγ Activity and the Antidiabetic Actions of Thiazolidinediones,” Cell 148:556-567 (2012), which are hereby incorporated by reference in their entirety). FGF1 is required for the remodeling of adipose tissue to adjust to fluctuations in nutrient availability (Jonker et al., “A PPARγ-FGF1 Axis is Required for Adaptive Adipose Remodelling and Metabolic Homeostasis,” Nature 485:391-394 (2012), which is hereby incorporated by reference in its entirety), and this process is influenced by FGF21 (Hotta et al., “Fibroblast Growth Factor 21 Regulates Lipolysis in White Adipose Tissue But is Not Required for Ketogenesis and Triglyceride Clearance in Liver,” Endocrinology 150:4625-4633 (2009); Dutchak et al., “Fibroblast Growth Factor-21 Regulates PPARγ Activity and the Antidiabetic Actions of Thiazolidinediones,” Cell 148:556-567 (2012), which are hereby incorporated by reference in their entirety). As part of a positive feedback loop, FGF21 stimulates PPARγ activity in adipocytes (Dutchak et al., “Fibroblast Growth Factor-21 Regulates PPARγ Activity and the Antidiabetic Actions of Thiazolidinediones,” Cell 148:556-567 (2012), which is hereby incorporated by reference in its entirety), raising the intriguing possibility that FGF21 regulates FGF1 signaling in adipose tissue through PPARγ. An FGF1ΔHBScore-FGF21C-tail chimera was generated in the same manner as the FGF2ΔHBScore-FGF21C-tail chimera (FIGS. 5 and 6). Specifically, K127, K128, and K133 of FGF1, which correspond to the key HS-binding residues identified in the crystal structure of heparin-bound FGF2 (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference in its entirety)), were mutated and then the short C-terminal tail of the mutated FGF1 was replaced with the C-terminal tail of FGF21 (P168 to S209) (FIG. 6). A full-length FGF1 protein harboring the HS-binding site mutations was used as a control (FIG. 6). Consistent with the structural prediction, this protein exhibited poor binding affinity for HS compared to wild-type FGF1 as evidenced by the fact that, unlike the wild-type ligand, the mutant protein did not bind to a Heparin sepharose column. A subcutaneous bolus injection of the FGF1ΔHBScore-FGF21C-tail chimera elicited a hypoglycemic effect in ob/ob mice (FIG. 9C), demonstrating that the chimera has metabolic activity. The effect was of similar magnitude as that observed for native FGF1 (FIG. 9C), which itself has a much greater hypoglycemic effect in ob/ob mice than native FGF21 (FIG. 9A). The HS-binding site mutant of FGF1, which was included as a control in these experiments, showed a similar hypoglycemic effect as the wild-type ligand (FIG. 9B), indicating that the loss in HS-binding affinity had no impact on the metabolic activity of FGF1. To alter the receptor-binding specificity of FGF1 such that FGF1 selectively binds to the “c” splice isoform of FGFR1, the principal receptor mediating the metabolic activity of FGF21, an N-terminally truncated FGF1 protein was made (FIG. 6). The truncated FGF1 ligand lacked twenty four residues from the N-terminus including the nine residues that are critical for the promiscuous binding of FGF1 to both splice isoforms of FGFR1-3 (Beenken et al., “Plasticity in Interactions of Fibroblast Growth Factor 1 (FGF1) N Terminus with FGF Receptors Underlies Promiscuity of FGF1,” J Biol Chem 287(5):3067-3078 (2012), which is hereby incorporated by reference in its entirety). Based on the crystal structures of FGF1-FGFR complexes, the truncation was also predicted to reduce the receptor-binding affinity of FGF1, and hence the ligand's mitogenicity. The truncated FGF1 protein induced a similar hypoglycemic effect in ob/ob mice as native FGF1 did (FIG. 9B), indicating that the metabolic activity of FGF1 is mediated through the “c” splice isoform of FGFR. Together, these findings provide a starting point for engineering FGF1 ligands that have no mitogenicity but the same or enhanced metabolic activity compared to native FGF1.
The demonstrated ability to convert a paracrine FGF into an endocrine ligand by means of reducing HS-binding affinity of the paracrine FGF and adding the Klotho co-receptor binding site substantiates that HS does not participate in the formation of the endocrine FGF signal transduction unit. The dispensability of HS for the metabolic activity of endocrine FGFs has an intriguing implication as to how these FGFs have evolved to become hormones. It appears that these ligands have lost the requirement to bind HS in order to signal, while acquiring the ability to bind Klotho co-receptors, which is necessary to direct these ligands to their target organs.
In the target tissue, Klotho co-receptors constitutively associate with cognate receptors of endocrine FGFs to offset the inherently low receptor-binding affinity of endocrine FGFs (FIGS. 10B-10D; Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007); Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Ogawa et al., Proc. Nat'l. Acad. Sci. U.S.A. 104:7432-7437 (2007); Urakawa et al., Nature 444:770-774 (2006), which are hereby incorporated by reference in their entirety). This low binding affinity is due to the fact that key receptor-binding residues in the β-trefoil core of endocrine FGFs are replaced by residues that are suboptimal for receptor binding (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporated by reference in its entirety). To measure the degree to which Klotho co-receptors enhance the receptor-binding affinity of endocrine FGFs, SPR experiments were conducted using FGF23 and FGFR1c and αKlotho co-receptor as an example (see FIGS. 10A-10F). The SPR data show that αKlotho enhances the affinity of FGF23 for FGFR1c by over 20-fold (FIGS. 10D and 10E). The affinity of FGF23 for FGFR1c in the presence of αKlotho is comparable to that of FGF2 for FGFR1c in the absence of its HS cofactor (FIGS. 10A and 10E). It should be noted, however, that HS further increases the binding affinity of FGF2 for FGFR1c by at least an order of magnitude (Pantoliano et al., Biochemistry 33:10229-10248 (1994); Roghani et al., J. Biol. Chem. 269:3976-3984 (1994), which are hereby incorporated by reference in their entirety). Hence, the receptor-binding affinity of FGF23 in the presence of αKlotho co-receptor still is lower than that of FGF2 in the presence of HS cofactor. These observations imply that the signaling capacity of the endocrine FGF signal transduction unit should be weaker than that of the paracrine FGF signaling unit. Indeed, cell-based studies show that even in the presence of their Klotho co-receptor, endocrine FGFs are inferior to paracrine FGFs at activating FGFR-induced intracellular signaling pathways (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007); Urakawa et al., Nature 444:770-774 (2006), which are hereby incorporated by reference in their entirety).
The finding that endocrine FGFs do not need to rely on HS for signaling has another important implication in regard to the role of Klotho co-receptors. Since FGFR dimerization is a prerequisite for FGF signaling in general, it is proposed that Klotho co-receptors not only enhance the binding affinity of endocrine ligand for receptor but also promote receptor dimerization upon ligand binding. In other words, Klotho co-receptors must fulfill the same dual role that HS plays in signaling by paracrine FGFs (FIG. 1D). The ligand conversion also provides the framework for the rational design of endocrine FGF-like molecules for the treatment of metabolic disorders. An FGF23-like molecule, for example, will be useful for the treatment of inherited or acquired hyperphosphatemia, and an FGF21-like molecule, for example, for the treatment of type 2 diabetes, obesity, and related metabolic disorders.
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

## Claims

1. A chimeric protein comprising:
an N-terminus coupled to a C-terminus, wherein the N-terminus comprises an FGF2 portion beginning at any one of residues 1 to 25 and ending at any one of residues 151-155 of SEQ ID NO: 121,
wherein the FGF2 amino acid positions corresponding to those selected from the group consisting of N36, K128, R129, K134, K138, Q143, K144, and combinations thereof are substituted to decrease binding affinity for heparin and/or heparan sulfate compared to FGF2 without the substitution, and
wherein the C-terminus comprises a C-terminal portion of an FGF23 comprising amino acid residues 161-251 of SEQ ID NO: 233.

2. The chimeric protein according to claim 1, wherein the FGF2 portion is amino acid residues 1-151 of SEQ ID NO: 121.

3. The chimeric protein according to claim 1, wherein the FGF2 portion is amino acid residues 25-151 of SEQ ID NO: 121.

4. The chimeric protein according to claim 1, wherein the FGF2 portion is amino acid residues 1-152, 1-153, 1-154, 1-155, 2-151, 2-152, 2-153, 2-154, 2-155, 3-151, 3-152, 3-153, 3-154, 3-155, 4-151, 4-152, 4-153, 4-154, 4-155, 5-151, 5-152, 5-153, 5-154, 5-155, 6-151, 6-152, 6-153, 6-154, 6-155, 7-151, 7-152, 7-153, 7-154, 7-155, 8-151, 8-152, 8-153, 8-154, 8-155, 9-151, 9-152, 9-153, 9-154, 9-155, 10-151, 10-152, 10-153, 10-154, 10-155, 11-151, 11-152, 11-153, 11-154, 11-155, 12-151, 12-152, 12-153, 12-154, 12-155, 13-151, 13-152, 13-153, 13-154, 13-155, 14-151, 14-152, 14-153, 14-154, 14-155, 15-151, 15-152, 15-153, 15-154, 15-155, 16-151, 16-152, 16-153, 16-154, 16-155, 17-151, 17-152, 17-153, 17-154, 17-155, 18-151, 18-152, 18-153, 18-154, 18-155, 19-151, 19-152, 19-153, 19-154, 19-155, 20-151, 20-152, 20-153, 20-154, 20-155, 21-151, 21-152, 21-153, 21-154, 21-155, 22-151, 22-152, 22-153, 22-154, 22-155, 23-151, 23-152, 23-153, 23-154, 23-155, 24-151, 24-152, 24-153, 24-154, 24-155, 25-152, 25-153, 25-154, or 25-155 of SEQ ID NO: 121.

5. The chimeric protein according to claim 1, wherein the one or more substitutions are selected from the group consisting of N36T; K128D; R129Q; K134V; K138H; Q143M; K144T, K144L, or K144I ; and combinations thereof.

6. A pharmaceutical composition comprising the chimeric protein according to claim 1 and a pharmaceutically-acceptable carrier.

7. The pharmaceutical composition according to claim 6 further comprising:
a hypophosphatemic agent, a phosphate binder, a vitamin D antagonist, an analgesic and/or an anti-inflammatory agent.

8. The chimeric protein according to claim 1, wherein the chimeric protein comprises the amino acid sequence of SEQ ID NO:323 or SEQ ID NO:324.

9. The chimeric protein according to claim 1, wherein the one or more amino acid substitutions comprises a substitution at amino acid residue N36.

10. The chimeric protein according to claim 9, wherein the substitution is N36T.

11. The chimeric protein according to claim 1, wherein the one or more amino acid substitutions comprises a substitution at amino acid residue K128.

12. The chimeric protein according to claim 11, wherein the substitution is K128D.

13. The chimeric protein according to claim 1, wherein the one or more amino acid substitutions comprises a substitution at amino acid residue R129.

14. The chimeric protein according to claim 13, wherein the substitution is R129Q.

15. The chimeric protein according to claim 1, wherein the one or more amino acid substitutions comprises a substitution at amino acid residue K134.

16. The chimeric protein according to claim 15, wherein the substitution is K134V.

17. The chimeric protein according to claim 1, wherein the one or more amino acid substitutions comprises a substitution at amino acid residue K138.

18. The chimeric protein according to claim 17, wherein the substitution is K138H.

19. The chimeric protein according to claim 1, wherein the one or more amino acid substitutions comprises a substitution at amino acid residue Q143.

20. The chimeric protein according to claim 19, wherein the substitution is Q143M.

21. The chimeric protein according to claim 1, wherein the one or more amino acid substitutions comprises a substitution at amino acid residue K144.

22. The chimeric protein according to claim 21, wherein the substitution is K144T.

23. The chimeric protein according to claim 21, wherein the substitution is K144L.

24. The chimeric protein according to claim 21, wherein the substitution is K144I.

25. The chimeric protein of claim 1, wherein the one or more amino acid substitutions comprises substitutions at amino acid residues K128, R129, and K134.

26. The chimeric protein of claim 25, wherein the substitutions are K128D, R129Q, and K134V.

27. A method for decreasing circulating levels of phosphate and/or bioactive vitamin D in a subject suffering from a disorder, the method comprising:
selecting a subject suffering from a disorder wherein the selected subject has a disorder associated with elevated blood levels of phosphate; elevated blood levels of bioactive vitamin D; or combinations thereof;
providing a chimeric protein according to claim 1; and
administering an amount of the chimeric protein to the selected subject effective to decrease circulating levels of phosphate and/or bioactive vitamin D in the selected subject.

28. The method according to claim 27, wherein the FGF2 portion is amino acid residues 1-151 of SEQ ID NO: 121.

29. The method according to claim 27, wherein the FGF2 portion is amino acid residues 25-151 of SEQ ID NO: 121.

30. The method according to claim 29, wherein the FGF2 portion is amino acid residues 1-152, 1-153, 1-154, 1-155, 2-151, 2-152, 2-153, 2-154, 2-155, 3-151, 3-152, 3-153, 3-154, 3-155, 4-151, 4-152, 4-153, 4-154, 4-155, 5-151, 5-152, 5-153, 5-154, 5-155, 6-151, 6-152, 6-153, 6-154, 6-155, 7-151, 7-152, 7-153, 7-154, 7-155, 8-151, 8-152, 8-153, 8-154, 8-155, 9-151, 9-152, 9-153, 9-154, 9-155, 10-151, 10-152, 10-153, 10-154, 10-155, 11-151, 11-152, 11-153, 11-154, 11-155, 12-151, 12-152, 12-153, 12-154, 12-155, 13-151, 13-152, 13-153, 13-154, 13-155, 14-151, 14-152, 14-153, 14-154, 14-155, 15-151, 15-152, 15-153, 15-154, 15-155, 16-151, 16-152, 16-153, 16-154, 16-155, 17-151, 17-152, 17-153, 17-154, 17-155, 18-151, 18-152, 18-153, 18-154, 18-155, 19-151, 19-152, 19-153, 19-154, 19-155, 20-151, 20-152, 20-153, 20-154, 21-155, 21-151, 21-152, 21-153, 21-154, 21-155, 22-151, 22-152, 22-153, 22-154, 22-155, 23-151, 23-152, 23-153, 23-154, 23-155, 24-151, 24-152, 24-153, 24-154, 24-155, 25-152, 25-153, 25-154, or 25-155 of SEQ ID NO: 121.

31. The method according to claim 27, wherein the one or more substitutions are selected from the group consisting of N36T; K128D; R129Q; K134V; K138H; Q143M; K144T, K144L, or K144I; and combinations thereof.

32. The method according to claim 27, wherein the administering is performed parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes.

33. The method according to claim 27, wherein the chimeric protein is administered with a pharmaceutically-acceptable carrier.

34. The method according to claim 27, wherein the selected subject is a mammal.

35. The method according to claim 27, wherein the selected subject is a human.

36. The method according to claim 27, wherein the chimeric protein is co-administered with a hypophosphatemic agent, a phosphate binder, a vitamin D antagonist, an analgesic and/or an anti-inflammatory agent.

37. The method according to claim 27, wherein the chimeric protein comprises the amino acid sequence of SEQ ID NO:323 or SEQ ID NO:324.

38. The method according to claim 27, wherein the selected subject has familial tumoral calcinosis.

39. The method according to claim 27, wherein the selected subject has hyperostosis-hyperphosphatemia syndrome.

40. The method according to claim 27, wherein the selected subject has hyperphosphatemia.