CHONDROITIN SULFATE SYNTHESIS PROMOTER

Abstract

The invention provides a chondroitin sulfate synthesis promoter useful for the treatment of diseases such as articular disease and discopathy. The chondroitin sulfate synthesis promoter contains, as an active ingredient, chondroitin sulfate glucuronyltransferase protein and/or chondroitin sulfate N-acetylgalactosaminyltransferase-1 protein, or a gene encoding the enzyme protein(s).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in situ hybridization results to confirm expression of chondroitin sulfate glycosyltransferases in cartilage (photographs);

FIG. 2 shows RT-PCR results representing expression patterns of chondroitin sulfate glycosyltransferases during differentiation of ATDC5 cells;

FIG. 3 shows RT-PCR results representing expression patterns of chondroitin sulfate glycosyltransferases during differentiation of N1511 cells;

FIG. 4 shows expression levels of chondroitin sulfate glycosyltransferases in ATDC5 cells, N1511 cells, and mouse embryonic fibroblasts (MEFs);

FIG. 5 shows expression levels of CSGlcAT and CSGalNAcT-1 in transfected cells;

FIG. 6 shows results of Alcian Blue staining and immunostaining (employing anti-chondroitin 4-sulfate (C4S) antibody or anti-aggrecan antibody) of control (Mock) and sample of transfected cells (photographs);

FIG. 7 shows chondroitin sulfate synthesis activities of control (Mock) and sample of transfected cells as determined by labeled chondroitin sulfate levels in culture media and cell lysates, wherein “-” denotes no treatment, “HSase” denotes heparitinase mixture treatment, and “CHase” denotes chondroitinase ABC treatment (lower graphs);

FIG. 8 shows results of disaccharide analysis of GAG digested products formed through chondroitinase ABC treatment of control (Mock) and sample of transfected cells, the disaccharide composition being determined in culture media and cell lysates;

FIG. 9 shows results of Alcian Blue staining of intervertebral disc in transgenic mice for CSGalNAcT-1 (photographs), wherein the photographs of a, c and c are for the mock, the photographs of b, d and f are for the transgenic mice, a and b are the overall view for the intervertebral disc, c and d are closeups for the nucleus pulposus cells and e and f are closeups for the chondrocytes; and

FIG. 10 shows levels for alcian blue stain of intervertebral disc in transgenic mice for CSGalNAcT-1.

Claims

1. A chondroitin sulfate synthesis promoter comprising, as an active ingredient, at least one of a gene encoding chondroitin sulfate glucuronyltransferase and a gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1.

2. A chondroitin sulfate synthesis promoter as described in claim 1, wherein the gene encoding chondroitin sulfate glucuronyltransferase is a nucleic acid molecule including a nucleotide sequence defined by SEQ ID NO: 1.

3. A chondroitin sulfate synthesis promoter as described in claim 1, wherein the gene encoding chondroitin sulfate glucuronyltransferase is a nucleic acid molecule which can be hybridized with a complement of a nucleic acid of SEQ ID NO: 1 under stringent conditions.

4. A chondroitin sulfate synthesis promoter as described in claim 1, wherein the gene encoding chondroitin sulfate glucuronyltransferase is a nucleic acid molecule including a nucleotide sequence identical to that defined by SEQ ID NO: 1, except that one or more nucleotide(s) have been substituted, deleted, inserted, or transposed, wherein the nucleic acid molecule encodes a protein having activity of transferring glucuronate from a glucuronate donor to a non-reducing end N-acetylgalactosamine residue present in the chondroitin skeleton.

5. A chondroitin sulfate synthesis promoter as described in claim 1, wherein the gene encoding chondroitin sulfate glucuronyltransferase is a nucleic acid molecule including a nucleotide sequence encoding an amino acid sequence defined by SEQ ID NO: 2.

6. A chondroitin sulfate synthesis promoter as described in claim 1, wherein the gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 is a nucleic acid molecule including a nucleotide sequence defined by SEQ ID NO: 3.

7. A chondroitin sulfate synthesis promoter as described in claim 1, wherein the gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 is a nucleic acid molecule which can be hybridized with a complement of a nucleic acid of SEQ ID NO: 3 under stringent conditions.

8. A chondroitin sulfate synthesis promoter as described in claim 1, wherein the gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 is a nucleic acid molecule including a nucleotide sequence identical to that defined by SEQ ID NO: 3, except that one or more nucleotide(s) have been substituted, deleted, inserted, or transposed, wherein the nucleic acid molecule encodes a protein having activity of transferring an N-acetylgalactosamine residue from an N-acetylgalactosamine donor to a non-reducing end D-glucuronate residue of an N-acetylgalactosamine acceptor substrate containing a sugar chain represented by formula; GlcUA-Gal-Gal-Xyl

9. A chondroitin sulfate synthesis promoter as described in claim 1, wherein the gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 is a nucleic acid molecule including a nucleotide sequence encoding an amino acid sequence defined by SEQ ID NO: 4.

10. A chondroitin sulfate synthesis promoter as described in claim 1, wherein at least one of a gene encoding chondroitin sulfate glucuronyltransferase and a gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 are incorporated into an expression vector.

11. A chondroitin sulfate synthesis promoter as described in claim 1, wherein at least one of a gene encoding chondroitin sulfate glucuronyltransferase and a gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 are incorporated into a host cell.

12. A chondroitin sulfate synthesis promoter comprising, as an active ingredient, at least one of a chondroitin sulfate glucuronyltransferase and a chondroitin sulfate N-acetylgalactosaminyltransferase-1.

13. A chondroitin sulfate synthesis promoter as described in claim 12, wherein the chondroitin sulfate glucuronyltransferase is an enzyme comprising a protein which includes an amino acid sequence defined by SEQ ID NO: 2.

14. A chondroitin sulfate synthesis promoter as described in claim 12, wherein the chondroitin sulfate glucuronyltransferase is an enzyme comprising a protein which includes an amino acid sequence identical to that defined by SEQ ID NO: 2, except that one or more amino acid residue(s) have been substituted, deleted, inserted, or transposed, wherein the protein has activity of transferring glucuronate from a glucuronate donor to a non-reducing end N-acetylgalactosamine residue present in the chondroitin skeleton.

15. A chondroitin sulfate synthesis promoter as described in claim 12, wherein the chondroitin sulfate N-acetylgalactosaminyltransferase-1 is an enzyme comprising a protein which includes an amino acid sequence defined by SEQ ID NO: 4.

16. A chondroitin sulfate synthesis promoter as described in claim 12, wherein the chondroitin sulfate N-acetylgalactosaminyltransferase-1 is an enzyme comprising a protein which includes an amino acid sequence identical to that defined by SEQ ID NO: 4, except that one or more amino acid residue(s)have been substituted, deleted, inserted, or transposed, wherein the protein has activity of transferring an N-acetylgalactosamine residue from an N-acetylgalactosamine donor to a non-reducing end D-glucuronate residue of an N-acetylgalactosamine acceptor substrate containing a sugar chain represented by formula; GlcUA-Gal-Gal-Xyl

17. A treatment agent for at least one of an articular disease and discopathy comprising, as an active ingredient, a chondroitin sulfate synthesis promoter comprising at least one of a chondroitin sulfate glucuronyltransferase, a gene encoding chondroitin sulfate glucuronyltransferase, a chondroitin sulfate N-acetylgalactosaminyltransferase-1, and a gene encoding a chondroitin sulfate N-acetylgalactosaminyltransferase-1.

18. A treatment agent for an articular disease as described in claim 17, which promotes synthesis of chondroitin sulfate in cartilage.

19. A cartilage repairing agent which can promote synthesis of chondroitin sulfate, the agent comprising, as an active ingredient at least one of a chondroitin sulfate glucuronyltransferase, a gene encoding chondroitin sulfate glucuronyltransferase, a chondroitin sulfate N-acetylgalactosaminyltransferase-1, and a gene encoding a chondroitin sulfate N-acetylgalactosaminyltransferase-1.

20. A cartilaginous tissue formation promoter comprising, as an active ingredient, at least one of a chondroitin sulfate glucuronyltransferase, a gene encoding chondroitin sulfate glucuronyltransferase, a chondroitin sulfate N-acetylgalactosaminyltransferase-1, and a gene encoding a chondroitin sulfate N-acetylgalactosaminyltransferase-1.

21. A method for promoting chondroitin sulfate synthesis in a living subject wherein the living subject includes a joint, a cartilage, an intervertebral disc or a cell, the method comprising the steps of: providing a gene comprising at least one of a gene encoding chondroitin sulfate glucuronyltransferase and a gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1; andintroducing the gene into the living subject.

22. A method for promoting a chondroitin sulfate synthesis in a living subject as described in claim 21, wherein the step of introducing the gene into the living subject is performed with a gene gun or via injection.

23. A method for promoting a chondroitin sulfate synthesis in a living subject wherein the living subject includes a joint, a cartilage, an intervertebral disc or a cell, the method comprising the steps of: providing at least one of chondroitin sulfate glucuronyltransferase and chondroitin sulfate N-acetylgalactosaminyltransferase-1; andintroducing at least one of a chondroitin sulfate glucuronyltransferase and chondroitin sulfate N-acetylgalactosaminyltransferase-1 into the living subject.

24. A method for promoting a chondroitin sulfate synthesis in a living subject as described in claim 23, wherein the step of introducing at least one of a chondroitin sulfate glucuronyltransferase and chondroitin sulfate N-acetylgalactosaminyltransferase-1 into the living subject is performed via injection.

25. A method for treating an articular disease or discopathy, the method comprising the steps of: applying a therapeutically effective amount of an active ingredient comprising at least one of a chondroitin sulfate glucuronyltransferase, a gene encoding chondroitin sulfate glucuronyltransferase, a chondroitin sulfate N-acetylgalactosaminyltransferase-1, and a gene encoding a chondroitin sulfate N-acetylgalactosaminyltransferase-1 with the affected joint or intervertebral disc; andpromoting a chondroitin sulfate synthesis in the affected joint or intervertebral disc.