WO2005095600A1 - ヘパラン硫酸糖鎖を付加したヘパリン結合性タンパク質、その製造方法及びそれを含有する医薬組成物 - Google Patents
ヘパラン硫酸糖鎖を付加したヘパリン結合性タンパク質、その製造方法及びそれを含有する医薬組成物 Download PDFInfo
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- WO2005095600A1 WO2005095600A1 PCT/JP2005/006364 JP2005006364W WO2005095600A1 WO 2005095600 A1 WO2005095600 A1 WO 2005095600A1 JP 2005006364 W JP2005006364 W JP 2005006364W WO 2005095600 A1 WO2005095600 A1 WO 2005095600A1
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- sugar chain
- heparan sulfate
- binding protein
- heparin
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Definitions
- the present invention relates to a heparin-binding protein that has been highly functionalized by selectively covalently binding a heparan sulfate sugar chain, a method for producing the same, and a pharmaceutical composition containing the same.
- heparin-binding proteins particularly proteins classified into the fibroblast growth factor (hereinafter referred to as "FGF") family, are sulfated polysaccharides such as heparin and heparan.
- FGF fibroblast growth factor
- heparin-binding protein such as fibroblast growth factor
- a sulfated polysaccharide such as henoline
- the biological activity and physical properties of the heparin-binding protein change, and the function changes. It has been known that the functions may be enhanced. However, even if the sulfated polysaccharide was mixed, the expected high functionality was limited. In addition, when these are used as pharmaceutical compositions, there has been a problem of undesired physiological activity due to free sulfated polysaccharide.
- Patent Document 1 "Glycosylated heparin-binding protein, method for producing the same, and No.3318602 (2002) Toru Imamura, Masahiro Asada, Osamu Oka, Osamu Suzuki, Atsuko Yoneda, Keiko Ota, Yuko Oda, Kazuko Takakawa, Noriko Orikasa, Tomoe Matsuda, Tetsuto Kojima : Yoneda A, Asada M, Oda Y, buzuki M, Imamura T. 'Engineering of an FGF— Proteoglycan Fusion Protein with Heparin— Independent, Deg radation- Augmented, Mitogenic Activity. "Nature Biotechnology 18 (6), 641-644 ( 2000)
- Non-Patent Document 2 Atsuko Yoneda, Masahiro Asada, Toru Imamura “Modification of Heparin-Binding Growth Factor FGF-1 Activity by Fusion with Syndecan” Cell Engineering 19 (9), 1338-1340 (2000) : Asaaa M, Yoneda A, Imamura T. Engineering of a
- An object of the present invention is to provide a heparan sulfate-selective glycosaminodalicanic sugar chain that is hardly containing chondroitin sulfate and covalently binds a glycosaminodalican sugar chain, with the aim of modifying the function of the hene binding protein.
- An object of the present invention is to establish a protein binding protein and a method for producing the same, and to provide a pharmaceutical composition containing the same.
- syndecan 4 sequence having a plurality of sites modified by both sugar chains of heparan sulfate and chondroitin sulfate.
- Analysis of the chimeric protein of the sugar chain modification site of syndecan 4 and the reporter revealed that the serine residue at position 39 in the primary structure (No. 9) was specifically modified by heparan sulfate.
- cDNA encoding a peptide capable of selectively receiving heparan sulfate (SEQ ID NO: 8) (SEQ ID NO: 13) and cDNA encoding a non-binding protein (SEQ ID NO: 7) (SEQ ID NO: 10) and the heparan sulfate sugar chain is covalently linked by producing the gene product of the linked cDNA in animal cells. It was found that the heterozygous binding protein can be produced by the combination. In addition, it was confirmed that the function of the henon-binding protein to which the heparan sulfate sugar chain was added was improved. The present invention has been completed based on such findings.
- the present invention provides a henolin-binding protein that has been highly functionalized by covalently binding a sugar chain rich in heparan sulfate.
- the henolin-binding protein of the present invention is a henolin-binding protein in which 90% or more of the composition is a heparan sulfate sugar chain, which is covalently linked to a sulfated glycosaminodalican sugar chain.
- the composition of the sulfated glycosaminodalican sugar chain can be determined, for example, according to the method described in Current Protocols in Molecular Biology, John Wiley & Sons, Inc., UNIT 17.13B (1996).
- the sugar chain is composed of (1) heparan sulfate, (2) an N-linked sugar chain combined with heparan sulfate and another type of glycosaminodalican, and (3) heparan sulfate and other glycosylate. It can be selected from the group consisting of O-linked sugar chains combined with Saminodalican, and (4) a combination thereof.
- the henolin binding protein may be a factor belonging to the FGF family or a closely related factor.
- the heparin-binding protein may have a sugar chain covalently bonded thereto via a peptide capable of preferentially receiving the addition of a heparan sulfate sugar chain.
- the holin-binding protein that covalently binds a sugar chain may be any one of the following (a) or (b):
- the amino acid sequence of SEQ ID NO: 6 or 7 has an amino acid sequence in which one or several amino acids have been deleted, substituted, added or modified, and has FGF activity; and A protein that can preferentially receive the addition of paran sulfate sugar chains.
- the FGF activity refers to an activity of promoting or suppressing the proliferation of fibroblasts, vascular endothelial cells, myoblasts, chondrocytes, osteoblasts, and glial cells. FGF activity can be measured according to the method described in Ornitz DM & Leder P., Journal of Biological Chemistry 267 (23), p. 16305-16311 (1992).
- Proteins that can preferentially receive the addition of heparan sulfate sugar chains include other sugars (eg, chondroitin) in vivo that have heparan sulfate sugar chain addition pathways. (Sulfated sugar chains) and can be received with higher selectivity. Just do it.
- Living organisms having a heparan sulfate sugar chain addition pathway include animal cells (eg, COS cells, CHO cells, BHK cells, NIH3T3 cells, BALB / c3T3 cells, HUVE cells, LEII cells), insect cells (eg, Sf-9 cells). , Tn cell), etc. The force is not limited to these.
- the present invention relates to a method for producing a non-binding protein having a composition in which 90% or more of the composition is a heparan sulfate sugar chain to which a sugar chain of glycosaminodalican sulfate is covalently bound.
- the peptide that can be preferentially subjected to the addition of heparan sulfate sugar chain may be any one of the following (a) or (b):!
- amino acids In the amino acid sequence of SEQ ID NO: 8, one or several amino acids may be deleted, substituted, added, or modified, and may preferentially receive heparan sulfate sugar chains. Peptides that can.
- Peptides that can preferentially receive the addition of heparan sulfate sugar chains can be used to convert the addition of heparan sulfate sugar chains to other saccharides (eg, chondroitin sulfate) in a living body having a heparan sulfate sugar chain addition pathway. Any substance that can be received with higher selectivity than the addition of a sugar chain) may be used.
- Living organisms having a heparan sulfate glycosylation pathway include animal cells (eg, COS cells, CHO cells, BHK cells, NIH3T3 cells, BALB / c3T3 cells, HUVE cells, LEII cells), and insect cells (eg, Sf-9 cell, Tn cell), etc. Will not be.
- animal cells eg, COS cells, CHO cells, BHK cells, NIH3T3 cells, BALB / c3T3 cells, HUVE cells, LEII cells
- insect cells eg, Sf-9 cell, Tn cell
- a cDNA encoding a peptide capable of receiving heparan sulfate sugar chain preferentially, a cDNA having the base sequence of SEQ ID NO: 13 can be mentioned.
- the heparin-binding protein is a heparin-binding protein in which 90% or more of the composition is a heparan sulfate sugar chain, to which a sugar chain of glycosaminodalican sulfate is covalently bonded.
- the sugar chains are (1) heparan sulfate, (2) N-linked sugar chains combined with heparan sulfate and other glycosaminodalicans, (3) heparan sulfate and other glycosaminoglycans. O-linked sugar chains, and (4) a combination thereof.
- the henolin binding protein may be a factor belonging to the FGF family or a closely related factor.
- Henoline-binding proteins have covalently linked sugar chains via peptides that can preferentially receive heparan sulfate sugar chains.
- the holin-binding protein that covalently bonds a sugar chain may be any one of the following (a) or (b):
- the present invention provides a pharmaceutical composition containing, as an active ingredient, the above-mentioned heparin-binding protein, which is highly functionalized by covalently binding a heparan sulfate sugar chain.
- the present invention provides a nucleic acid that encodes a peptide having the base sequence of SEQ ID NO: 13 and capable of preferentially receiving carohydrate of a heparan sulfate sugar chain.
- the nucleic acid include DNA, RNA, chimeric molecules of DNA and RNA, derivatives thereof, and the like.
- the nucleic acid is Preferably, it is DNA.
- the term "sulfuric acid glycosaminodalican sugar chain” refers to a variety of sugars that extend from xylose bound to a serine residue in the primary structure of a protein or exist in a free state. It is a generic term for chain structures, such as N-acetyldarcosamine and N- It has a repeating structure consisting of an amino sugar represented by acetyl galatatosamine and a disaccharide unit represented by diuronic acid or galactosca represented by glucuronic acid and diuronic acid.Some of the hydroxyl groups or amino groups are replaced by sulfate groups. What is being said.
- a heparin-binding protein that selectively covalently binds a heparan sulfate sugar chain and contains almost no chondroitin sulfate, and a method for producing the same are provided.
- the heparin-binding protein of the present invention has a higher degree of functional modification than a heparin-binding protein containing heparan sulfate and chondroitin sulfate.
- the heparin-binding protein of the present invention can be used as a pharmaceutical.
- FIG. 1 shows that trunc. PG-FGF-l protein secreted by COS-1 cells was separated by SDS denaturing electrophoresis and stained with anti-FGF-1 monoclonal antibody in Test Example 1. 4 is an electrophoresis photograph showing the results.
- FIG. 2 shows that in Test Example 2, trunc. PG-FGF-1 protein and S / FGF-la-II protein were converted to various glycosaminodalicanases (GAG'ase) and peptide N-glycosidase in Test Example 2.
- GAG'ase glycosaminodalicanases
- 5 is an electrophoretic photograph showing the results of analysis by SDS denaturing electrophoresis and anti-FGF-1 monoclonal antibody staining after treatment with F.
- FIG. 3 is a graph showing the results of evaluating the cell growth promoting activity of Ba / F3 cells expressing FGF receptors of various proteins in Test Example 3.
- the henon-binding protein to which a heparan sulfate sugar chain is to be covalently bound is a protein having a henoline binding property, and as a specific example, belongs to the FGF family. Or closely related factors, or those that have heparin binding but have no structural similarity to the former! /, And other proteins. Other proteins described here include, specifically, heparin-binding epidermal growth factor-like factor (HB-EGF), platelet-derived growth factor (PDGF), and the like. Absent. Specific examples of factors belonging to the FGF family include FGF-1 to 23.
- the heparin-binding protein may be covalently bound to a heparan sulfate sugar chain via a peptide capable of receiving sugar chain addition.
- the heparin-binding protein for covalently binding a heparan sulfate sugar chain may be any of the following proteins (a) or (b).
- the protein having the amino acid sequence of SEQ ID NO: 7 is encoded by, for example, the DNA sequence of SEQ ID NO: 10.
- the heparin-binding protein for covalently binding the heparan sulfate sugar chain may be any of the following proteins (a,) or (b,). (a ′) a protein consisting of the amino acid sequence of SEQ ID NO: 6;
- the protein having the amino acid sequence of SEQ ID NO: 6 is encoded by, for example, the DNA sequence of SEQ ID NO: 5.
- This protein contains, in addition to the peptide sequence of a factor belonging to the FGF family, a peptide sequence capable of preferentially receiving the addition of heparan sulfate sugar chain and a signal peptide sequence.
- the term "heparin-binding protein” refers to a secretory protein called a signal peptide existing at the amino terminus when the cDNA described in the sequence listing is added to the protein defined below and secreted from the cell. Includes truncated forms of the peptide sequence.
- the henolin-binding protein contained as an active ingredient of the pharmaceutical composition of the present invention is
- the heparan sulfate sugar chain to be covalently bound to the heparin-binding protein may be any one as long as the heparin-binding protein is highly functionalized by covalently binding.
- “highly functional” means that the activity of the target protein is increased.
- the heparan sulfate sugar chain is covalently bound to the protein, and the active force remaining after the treatment with heat, acid or alkali. In comparison, it is higher.
- the heparan sulfate sugar chain refers to the above-mentioned sulfated glycosaminodalican sugar chain, in which the amino sugar is N-acetyldarcosamine, and a peronic acid (glucuronic acid or iduronic acid). Having a repeating structure of a disaccharide unit, wherein some of the hydroxyl groups or amino groups are substituted with sulfate groups.
- This heparan sulfate sugar chain may have an addition, deletion, substitution or modification in a part of the sugar chain sequence as long as it exerts its function.
- heparan sulfate sugar chain-added henon-binding protein In binding a heparan sulfate sugar chain to a heparin-binding protein, only the heparan sulfate sugar chain may be directly covalently bound to the heparin-binding protein.
- a peptide of any length covalently linked to a sulfated sugar chain may be covalently linked to the hetero-binding protein.
- heparan sulfate sugar chain-added henon-binding protein To produce the heparan sulfate sugar chain covalently bound heparan sulfate protein of the present invention (hereinafter referred to as "heparan sulfate sugar chain-added henon-binding protein").
- a cDNA encoding a peptide capable of preferentially receiving addition of a heparan sulfate sugar chain is linked to a cDNA encoding a heparin-binding protein, and this is incorporated into an appropriate expression vector. May be introduced into a host cell having a glycosylation pathway to express a heparan sulfate glycosylation type heparin-binding protein.
- cDNAs of various heparin-binding proteins are designed by appropriate primers from sequences registered in gene banks such as DDBJ (Japan DNA Data Bank), and RT-PCR (RT-PCR) is performed from mRNA of the relevant tissue of the animal. It can be obtained by performing reverse transcription PCR).
- DDBJ Joint DNA Data Bank
- RT-PCR RT-PCR
- Peptides that are known to preferentially receive heparan sulfate sugar chains include core proteins of various proteodaricans (eg, syndecan, glypican, perlecan, etc.) or a part thereof. . As a part of the core protein of proteodarican, it contains Ser-Gly repeat sequence which is considered to be a glycosylation site of proteodalican. Peptides. Examples of peptides that can receive heparan sulfate sugar chain preferentially include the following peptides (a) and (b).
- amino acids In the amino acid sequence of SEQ ID NO: 8, one or several amino acids may be deleted, substituted, added, or modified, and may preferentially receive heparan sulfate sugar chains. Peptides that can.
- the site to which the heparan sulfate sugar chain is bound may be a site where a turn is formed in the secondary structure of the henon-binding protein, or the vicinity of the terminal or a three-dimensional structure due to sugar chain-attached sugar. The part does not change significantly.
- an oligonucleotide encoding a peptide known to preferentially receive a secretion signal and the addition of heparan sulfate sugar chain is synthesized or amplified by a PCR reaction, and this is amplified by a heline binding property. Insert at the 5 'end of the plasmid encoding the protein.
- the secretory signal peptide for example, the amino terminus of a typical secretory glycoprotein can be used, and specific examples include amino acids 18 residues from the N-terminus of human syndecan-14.
- a plasmid encoding a heparin binding protein can be prepared by incorporating DNA encoding a heparin binding protein into an appropriate plasmid. This
- any plasmid can be used as long as it can be replicated and maintained in the host.
- pBR322 and pUC18 derived from Escherichia coli, and constructed based on these, PET-3c and the like.
- Examples of a method for incorporating the above-mentioned oligonucleotide into a plasmid encoding a heparin-binding protein include the method described in T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p. 239 (1982).
- a secretion signal From the plasmid prepared as described above, a secretion signal, bases encoding all peptides of heparin-binding protein known to preferentially receive carohydrate attached to heparan sulfate sugar chains, and bases encoding all of heparin-binding proteins Region containing the sequence (hereinafter referred to as "heparan sulfate Region containing a nucleotide sequence encoding a compatible protein ". ) Is cut out and ligated downstream of a promoter in a vector suitable for expression, whereby an expression type vector can be obtained.
- the region containing the nucleotide sequence encoding the glycosylated heme-binding protein has ATG at the 5 'end as a translation initiation codon, and TAA and TGA at the 3' end as translation termination codons. Or it may have a TAG.
- a promoter is connected upstream thereof.
- the promoter used in the present invention may be any promoter that is suitable for the host used for gene expression. When the host to be transformed is an animal cell, examples include an SV40-derived promoter and a retrovirus promoter.
- any plasmid that can be expressed in host cells should be used.
- a vector constructed based on pBR322, PUC18 derived from Escherichia coli and the like can be mentioned.
- Examples of the method for incorporation into a plasmid include the method described in T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p. 239 (1982).
- a transformant carrying the vector is produced.
- Any host cell can be used as long as it has a heparan sulfate sugar chain addition pathway (eg, COS cell, CHO cell, BHK cell, NIH3T3 cell, BALB / c3T3 cell, HUVE cell). , LEII cell), insect cells (eg, Sf-9 cell, Tn cell) and the like. The present invention is not limited to these.
- the above transformation is performed by a method generally performed for each host.
- any method that is not general but applicable can be used.
- the host is an animal cell
- a vector containing the recombinant DNA is introduced into cells in the growth phase or the like by the calcium phosphate method, the lipofection method, or the electoral poration method.
- a heparan sulfate sugar chain-added heparin-binding protein is produced.
- the medium used for culturing is generally used for each host. Is used. Alternatively, any medium that is not general but can be used may be used.
- the host is an animal cell, one obtained by adding animal serum to Dulbecco's MEM or the like is used. Culture is performed under conditions generally used for each host. In addition, it is sufficient if the conditions are not general but applicable. For example, if the host is animal cells, perform at about 32 to 37 ° C, 5% C02, 100% humidity for about 24 hours to 2 weeks, and change the gas phase conditions or add agitation as necessary. Can be.
- the protein released into the culture is separated from the supernatant after centrifugation. Can be collected directly.
- the supernatant fluid can be purified by appropriately combining known separation and purification methods to purify the heparan sulfate sugar chain-added henon-binding protein.
- known separation and purification methods include salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis, ion exchange chromatography, affinity chromatography, reverse phase High performance liquid chromatography, isoelectric focusing and the like can be used.
- the preparation thus obtained can be dialyzed and freeze-dried to obtain a dry powder as long as the activity of the heparan sulfate sugar chain-added heptane-binding protein is not impaired. Further, adding and storing serum albumin or the like as a carrier is effective for preventing the sample from adsorbing to the container. Further, the coexistence of a trace amount of a reducing agent in the purification step or the storage step is suitable for preventing oxidation of the sample.
- the reducing agent include ⁇ -mercaptoethanol, dithiothreitol, and glutatin.
- glycosylated heparin-binding protein of the present invention can also be produced by binding a sugar chain to a heparin-binding protein by a chemical method.
- a chemical method one of the following a) and b) or a combination of these methods can be considered.
- a heparan sulfate sugar chain is first completed by a biological method, a chemical synthesis method, or a combination thereof.
- an appropriate protein-binding residue may be introduced into the sugar chain terminal.
- an aldehyde group is formed, and this is amino-bonded to an amino group in the protein, thereby completing the binding of the heparan sulfate sugar chain to the protein. I do.
- an aldehyde group is formed by reducing and partially oxidizing a reducing end of a monosaccharide or an appropriate protein-binding residue bonded to the monosaccharide, and this is converted to an amino acid in a protein.
- the amino bond to the group completes the bond between the monosaccharide and the protein.
- a hetero sulfate sugar chain is completed. This binding may be a biological method, a chemical synthesis method, or a method of appropriately combining them.
- a protein that has been highly functionalized by covalently binding a heparan sulfate sugar chain to a henolin-binding protein can be used as a medicine.
- the heparan sulfate sugar chain-added heparin-binding protein of the present invention has an effect of regulating the physiological function of FGF.
- the physiological function of FGF refers to promoting or suppressing the proliferation of fibroblasts, vascular endothelial cells, myoblasts, chondrocytes, osteoblasts, and glial cells.
- the heparan sulfate sugar chain-added henon-binding protein of the present invention is effective for promoting cell proliferation and regeneration of tissues such as liver, controlling wound healing and nervous function, and regulating proliferation of fibroblasts and the like.
- Various diseases specifically, fibroblastoma, hemangiomas, osteoblastoma, neuronal cell death, Alheimer's disease, Parkinson's disease, neuroblastoma, amnesia, dementia, prevention and cure of myocardial infarction It is useful for treatment, and can also be used as a hair growth agent, a hair restorer, and the like.
- the heparan sulfate sugar chain-added heparin-binding protein of the present invention can be prepared by using a pharmaceutically acceptable solvent, excipient, carrier, adjuvant, etc., in the form of a liquid preparation, a batch preparation, or the like, according to a standard method for preparation of a pharmaceutical preparation.
- Pharmaceutical compositions such as screens, aerosols, injections, powders, granules, tablets, suppositories, enteric agents and capsules may be used.
- the content of the heparan sulfate sugar chain-added heparin-binding protein as an active ingredient in the pharmaceutical composition may be about 0.0000000001 to 1.0% by weight.
- the pharmaceutical composition can be safely administered parenterally or orally to mammals such as humans, mice, rats, puppies, dogs and cats.
- the dose of the pharmaceutical composition may be appropriately changed depending on the dosage form, administration route, symptoms, and the like.
- the heparin-sulfated sugar chain-added heparin-binding protein is administered.
- the quality is exemplified by applying 0.0001-100 mg to the affected area several times a day.
- a natural protein having no sugar chains other than the henolin-binding protein can be obtained by covalently linking the sugar chains. Can also be enhanced.
- phR7A8 is a plasmid in which the human ryucan cDNA (PCR product) is inserted into the EcoR V site of pBluescript II (KS +) cloning vector. Including the 7th to 2610th of the mRNA sequence shown in Accession No. D13292 (see B.B.R.C.Vol. 190, No. 3, p. 814-822, 1993). This was digested with Pvu II, and the obtained DNA fragment of 2,232 base pairs was subjected to PCR (Polymerase Chain Reaction) using the ⁇ type.
- PCR Polymerase Chain Reaction
- the resulting 422 base pair band was separated and extracted, and inserted into pBluescript II (KS +) cloning vector (2934 base pairs) double-cut with EcoR I and Sal I to obtain FGF-la. / pBluescript II (KS +) was obtained.
- FGF-la / pBluescript II (KS +) was sequentially digested with Aor51H I and Sal I, and the resulting 2626 base pair band was separated and extracted, and used for the ligation reaction described below.
- PCR polymerase chain reaction
- # 117 5, -tcttccgatagactgcgtcg- «3,) (Rooster system No. 1) and # RI45 (5-gtaattagctacatcctcatcgtctgg-3 ') (SEQ ID NO: 2) were used.
- the specifically amplified 200 bp band was separated by electrophoresis and extracted.
- the DNA sequence of the translation region of the S / FGF-la-11 / pMEXneo plasmid is shown in SEQ ID NO: 11. It encodes a protein having the amino acid sequence of SEQ ID NO: 12 in mammalian cells.
- PCR was performed using the S / FGF-la-II / pMEXneo plasmid as type III.
- # 646 ⁇ -gaggatgtagctaattacaagaagccca-3,) (rooster sequence number 3) and # 118 (5-cattctagttgtggtttgtcc-3 ') (sequence number 4) were used. 479 base pairs specifically amplified was separated by electrophoresis and extracted.
- the DNA fragments obtained in the above (1) and (2) were mixed to form ⁇ , and PCR was performed using # 117 and # 118 as primers.
- the specifically amplified 661 bp band was separated by electrophoresis and extracted. This was further cut with EcoR I and Sal I, and the resulting 564 bp band was separated and extracted. This was inserted into a pMEXneo expression vector (5916 base pairs) double-cut with EcoR I and Sal I to obtain trunc.
- PG-FGF-1 / pMEXneo This expression vector contains the nucleotide sequence of SEQ ID NO: 5, which encodes a protein having the amino acid sequence of SEQ ID NO: 6 in mammalian cells.
- the obtained trunc. PG-FGF-1 / pMEXneo was transfected into COS-1 cells (monkey kidney-derived cell line) by the lipofection method. By culturing in a serum-free medium, the protein synthesized in the medium was secreted, and the culture supernatant was collected 3 days later. The obtained conditioned medium was centrifuged at low speed, and the supernatant was stored at 4 ° C.
- the conditioned medium of trunc. PG-FGF-1 protein secreting cells was dialyzed against distilled water and concentrated by freeze-drying. Add this to the electrophoresis buffer (containing SDS, 2-mercaptoethanol). Boiled together to prepare a sample for electrophoresis. Perform electrophoresis using 12.5% acrylamide gel in the presence of SDS and 2-mercaptoethanol.Transfer electrically to nitrocellulose membrane, anti-FGF-1 monoclonal antibody, horseradish peroxidase-labeled anti-mouse. The cells were stained with an IgG antibody and detected by a chemiluminescence method. The results are shown in Figure 1.
- the left line shows the migration position of the standard protein with a known molecular weight and its molecular weight (unit: dalton).
- the migration position of the molecular weight marker is shown on the left.
- PG-FGF-1 protein is a protein with 183 amino acid residues as shown in SEQ ID NO: 6. It is expressed as an unmodified simple protein.If secreted, it should give a band around 20 kDa. It is. However, it was actually detected as a smear band around 40 to 100 kDa. This is a characteristic pattern for proteins modified with glycosaminodalican sugar chains. It is considered that the smear is caused by the uneven length of glycosaminodalican sugar chains.
- PG-FGF-1 treated with a mixture of heparanase and heparitinase HP, ase & HS, ase
- HP, ase & HS, ase completely eliminates the smear band and gives two bands at 21 kDa and 25 kDa.
- S / FGF-la-II is a mixture of heparanase and heparitinase (HP 'ase &HS' ase) alone, and although some of the molecules are reduced in molecular weight, it is not possible to completely eliminate the smear band.
- the physiological activity of trunc. PG-FGF-1 protein as a growth factor was evaluated.
- FGF-1 binds to a target cell receptor and exerts its biological activity, coexistence of heparin or heparan sulfate is essential. Therefore, we targeted a cell line that expressed the FGF receptor (Rlc type) on mouse Ba / F3 cells (a cell line derived from pro B cells), which are known not to express endogenous heparan sulfate.
- the cell growth promoting activity of trunc. PG-FGF-1 protein was evaluated in the presence or absence of heparin. The evaluation of the cell growth promoting activity was based on the measurement of the number of living cells using Tetracolor One.
- Ba / F3 cells expressing the FGF receptor were seeded on a 96-well plate, and at the same time, crudely purified trunc. PG-FGF-1 protein was added at various concentrations. At this time, 10 ug (microgram) co-existing and non-co-existing heparin were prepared. Forty-eight hours later, Tetracolor One was added with 10 ⁇ l (micro-liter) of casket, and the mixture was kept warm for 4 hours, and then the absorbance at 450 nm was measured.
- the cell growth promoting activity is represented by the ratio of the absorbance at 450 nm in the presence of each growth factor, with the absorbance at 450 nm when the cells are cultured in a growth medium (containing IL-3) of Ba / F3 cells.
- the S / FGF-la-II protein shows activity at high concentrations (10-100 ng / ml) even without heparin (triangular dashed line).
- PG-FGF-1 protein exhibits activity even at a low concentration of 1 to 10 ng / ml without coexisting with henolin (circled line). Its activity is comparable to the case where heparin coexists with simple protein FGF-1 (solid line) and has a higher specific activity than S / FGF-la-II. Based on the above results, the S / FGF-la-II protein and trunc.
- PG-FGF-1 protein have a heparan sulfate sugar chain in their own molecules (see Fig. 2), which substitutes for the function of heparin. It is thought that it is.
- the S / FGF-la-II protein has not only a heparan sulfate sugar chain but also a chondroitin sulfate sugar chain, which is thought to contribute to exerting its activity. Specific activity is not very high.
- PG-FGF-1 protein is almost exclusively modified with heparan sulfate sugar chain, it is considered that high specific activity was obtained.
- the novel heparan sulfate sugar chain-added henon-binding protein of the present invention has excellent stability such as heat resistance, acid resistance, alkali resistance, and protease resistance, and has high biological activity. Therefore, the use of the heparan sulfate sugar chain-added heparin-binding protein of the present invention in pharmaceuticals provides excellent in vivo stability, especially acid resistance, alkali resistance, and immature stability. Drugs that can be applied to medicines and have high medicinal effects can be designed.
- SEQ ID NO: 1 shows the nucleotide sequence of primer # 117 used in Example 1.
- SEQ ID NO: 2 shows the nucleotide sequence of primer # 645 used in Example 1.
- SEQ ID NO: 3 shows the nucleotide sequence of primer # 646 used in Example 1.
- SEQ ID NO: 4 shows the nucleotide sequence of primer # 118 used in Example 1.
- SEQ ID NO: 5 shows the nucleotide sequence of trunc. PG-FGF-1.
- SEQ ID NO: 6 shows the amino acid sequence of trunc. PG-FGF-1.
- SEQ ID NO: 7 shows the amino acid sequence of FGF-la.
- SEQ ID NO: 8 shows a partial amino acid sequence of human syndecan-4.
- SEQ ID NO: 9 shows the entire amino acid sequence of human syndecan-4.
- SEQ ID NO: 10 shows the nucleotide sequence of FGF-la.
- SEQ ID NO: 11 shows the nucleotide sequence of S / FGF-la-II.
- SEQ ID NO: 12 shows the amino acid sequence of S / FGF-la-II.
- SEQ ID NO: 13 shows a partial nucleotide sequence of human syndecan-4.
- SEQ ID NO: 14 shows the entire nucleotide sequence of human syndecan-4.
- SEQ ID NO: 15 shows the nucleotide sequence of primer # 109 used in Reference Example 1.
- SEQ ID NO: 16 shows the nucleotide sequence of primer # 111 used in Reference Example 1.
- SEQ ID NO: 17 shows the nucleotide sequence of primer # 967 used in Reference Example 1.
- SEQ ID NO: 18> shows the nucleotide sequence of primer # 967 used in Reference Example 1.
- SEQ ID NO: 18 shows the nucleotide sequence of primer # 630 used in Reference Example 1.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB0621466A GB2427863B (en) | 2004-03-31 | 2005-03-31 | Heparin-Binding Protein Having Heparan Sulfate Sugar Chain Attached Thereto Process For Producing The Same And Medicinal Composition Containing The Same |
DE112005000737T DE112005000737T5 (de) | 2004-03-31 | 2005-03-31 | Mit Heparansulfat-Zuckerketten modifizierte Heparin-bindende Proteine, Verfahren zur Erzeugung derselben und Arzneimittel, welche dieselben enthalten |
US11/547,346 US7741078B2 (en) | 2004-03-31 | 2005-03-31 | Heparin-binding protein modified with heparan sulfate sugar chains, process for producing the same and pharmaceutical compositions containing the same |
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JP2004108570A JP4505631B2 (ja) | 2004-03-31 | 2004-03-31 | ヘパラン硫酸糖鎖を付加したヘパリン結合性タンパク質、その製造方法及びそれを含有する医薬組成物 |
JP2004-108570 | 2004-03-31 |
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WO2005095600A1 true WO2005095600A1 (ja) | 2005-10-13 |
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PCT/JP2005/006364 WO2005095600A1 (ja) | 2004-03-31 | 2005-03-31 | ヘパラン硫酸糖鎖を付加したヘパリン結合性タンパク質、その製造方法及びそれを含有する医薬組成物 |
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US (1) | US7741078B2 (ja) |
JP (1) | JP4505631B2 (ja) |
DE (1) | DE112005000737T5 (ja) |
GB (1) | GB2427863B (ja) |
WO (1) | WO2005095600A1 (ja) |
Cited By (2)
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JP2010530856A (ja) * | 2007-06-22 | 2010-09-16 | シュー,ハンメイ | 血管生成の抑制剤、並びにその製造方法、修飾方法及び抗腫瘍薬の製造における用途 |
US7956033B2 (en) * | 2008-06-10 | 2011-06-07 | Eu Sol Biotech Co., Ltd. | Modified peptide of human acidic fibroblast growth factor |
Families Citing this family (12)
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JOP20190083A1 (ar) | 2008-06-04 | 2017-06-16 | Amgen Inc | بولي ببتيدات اندماجية طافرة لـfgf21 واستخداماتها |
PE20120021A1 (es) | 2008-10-10 | 2012-02-10 | Amgen Inc | Mutantes fgf21 |
CA2760674A1 (en) | 2009-05-05 | 2010-11-11 | Amgen Inc. | Fgf21 mutants and uses thereof |
NZ596037A (en) | 2009-05-05 | 2013-10-25 | Amgen Inc | Fgf21 mutants and uses thereof |
EP2443145A1 (en) * | 2009-06-17 | 2012-04-25 | Amgen, Inc | Chimeric fgf19 polypeptides and uses thereof |
TWI434698B (zh) * | 2009-08-14 | 2014-04-21 | Eu Sol Biotech Co Ltd | 人類酸性纖維母細胞生長因子之醫藥用途 |
WO2011028668A2 (en) * | 2009-09-01 | 2011-03-10 | Zhenyu Wang | K5 heparosan fermentation and purification |
CA2782814A1 (en) * | 2009-12-02 | 2011-06-09 | Amgen Inc. | Binding proteins that bind to human fgfr1c, human .beta.-klotho and both human fgfr1c and human .beta.-klotho |
EP2338909B1 (en) | 2009-12-08 | 2013-07-10 | EU Sol Biotech Co., Ltd. | Modified peptide of human acidic fibroblast growth factor |
JP2011121882A (ja) * | 2009-12-09 | 2011-06-23 | Eu Sol Biotech Co Ltd | ヒト酸性繊維芽細胞増殖因子の改変ペプチド |
US9517264B2 (en) | 2010-04-15 | 2016-12-13 | Amgen Inc. | Human FGF receptor and β-Klotho binding proteins |
JP2014193870A (ja) * | 2014-04-23 | 2014-10-09 | Eu Sol Biotech Co Ltd | ヒト酸性繊維芽細胞増殖因子の改変ペプチド |
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JPH11137255A (ja) * | 1997-11-10 | 1999-05-25 | Agency Of Ind Science & Technol | 糖鎖付加型ヘパリン結合性タンパク質、その製造方法およびそれを含有する医薬組成物 |
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US5486599A (en) * | 1989-03-29 | 1996-01-23 | The Board Of Trustees Of The Leland Stanford Junior University | Construction and use of synthetic constructs encoding syndecan |
US5622562A (en) * | 1993-05-27 | 1997-04-22 | Alcan International Limited | Coating strip material with protective decorative layers while avoiding use of solvents |
IL133318A0 (en) * | 1999-12-05 | 2001-04-30 | Yeda Res & Dev | Proteoglycans and pharmaceutical compositions comprising them |
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JPH11137255A (ja) * | 1997-11-10 | 1999-05-25 | Agency Of Ind Science & Technol | 糖鎖付加型ヘパリン結合性タンパク質、その製造方法およびそれを含有する医薬組成物 |
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ASADA M. ET AL: "Engineering of a Heparin-Binding Growth Factor with Heparan Sulfate Sugar Chains.", TRENDS GLYCOSCIENCE GLYCOTECHNOLOGY., vol. 13, no. 72, 2001, pages 385 - 394, XP002993406 * |
ASADA M. ET AL: "Modification of FGF-1, preferentially with heparan sulfate but not chondroitin sulfate.", vol. 76, no. 8, 25 August 2004 (2004-08-25), pages 1006, XP002993405 * |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010530856A (ja) * | 2007-06-22 | 2010-09-16 | シュー,ハンメイ | 血管生成の抑制剤、並びにその製造方法、修飾方法及び抗腫瘍薬の製造における用途 |
US7956033B2 (en) * | 2008-06-10 | 2011-06-07 | Eu Sol Biotech Co., Ltd. | Modified peptide of human acidic fibroblast growth factor |
Also Published As
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JP2005287431A (ja) | 2005-10-20 |
GB0621466D0 (en) | 2006-12-20 |
DE112005000737T5 (de) | 2007-04-12 |
GB2427863B (en) | 2008-12-17 |
US7741078B2 (en) | 2010-06-22 |
JP4505631B2 (ja) | 2010-07-21 |
US20080119403A1 (en) | 2008-05-22 |
GB2427863A (en) | 2007-01-10 |
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