Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-06-23
2002-04-16
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S183000
Reexamination Certificate
active
06372466
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a novel sulfotransferase and more particularly to a galactosaminoglycan sulfotransferase which has an activity of transferring a sulfate group from a sulfate group donor to a hydroxyl group at the C-4 position of a galactosamine residue contained in galactosaminoglycan.
Glycosaminoglycan is a polysaccharide which has a disaccharide unit consisting of an aminosugar residue and an uronic acid residue as a basic skeleton. Such glycosaminoglycan includes, for example, hyaluronic acid, chondroitin, chondroitin 4-sulfate (chondroitin sulfate A), chondroitin 6-sulfate (chondroitin sulfate C), dermatan sulfate (chondroitin sulfate B), keratan sulfate, heparan sulfate and heparin. Of these, those glycosaminoglycans which have a galactosamine residue as the aminosugar residue are called galactosaminoglycan, in which chondroitin, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate and the like are classified.
Recently, various physiological functions of glycosaminoglycans have attracted attention.
Particularly, it has been being gradually clarified that the amount and position, for example, of sulfate groups which they have are associated with such physiological functions. Thus, the change in physiological activity of glycosaminoglycan due to the change in its sulfate group suggests the possibility that glycosaminoglycan and modifications thereof will be useful as a medicine. Accordingly, research for a sulfotransferase associated with the change in a sulfate group has been promoted, which results in the knowledge of various enzymes which transfer a sulfate group to a specified position of the basic skeleton of glycosaminoglycan, and utilization of such enzymes in the modification of glycosaminoglycan is expected.
Such glycosaminoglycan sulfotransferase includes, for example, chondroitin 6-sulfotransferase (C6ST: transfers a sulfate group from a sulfate group donor to the C-6 position of galactosamine residue; Habuchi, O., Matsui, Y., Kotoya, Y., Aoyama, Y., Yasuda, Y., and Noda, M., (1993) J. Biol. Chem. 268, 21968-21974), haparan sulfate 2-O-sulfotransferase (HS2ST: transfers a sulfate group from a sulfate group donor to the C-2 position of uronic acid residue; Japanese Patent Application Laid-open No. 9-28374 (1997)), heparan sulfate 6-O-sulfotransferase (HS6ST: transfers a sulfate group from a sulfate group donor to the C-6 position of glucosamine residue; Japanese Patent Application Laid-open No. 8-33483 (1996)), and keratan sulfate 6-O-sulfotransferase (KSGal6ST: transfers a sulfate group to the C-6 position of galactose residue; Fukuta, M., Inazawa, J., Torii, T., Tsuzuki, K., Shimada, E., and Habuchi, O. (1997) J. Biol. Chem. 272(51), 32321-32328). However, no enzyme has been isolated that has an activity of transferring a sulfate group to the C-4 position of galactosamine residue of glycosaminoglycan having a galactosamine residue (galactosaminoglycan).
SUMMARY OF THE INVENTION
If only a specified position of the basic skeleton of galactosaminoglycan can be sulfated, then it might be possible to create new galactosaminoglycan that has a physiological activity. It is difficult to sulfate only a specified position by a chemical technique and hence utilization of enzymes is suitable for such a purpose. However, it is only C6ST that has been known as the purified enzyme which can sulfate galactosaminoglycan, so that enzymes that have an activity of transferring a sulfate group to the hydroxyl group at the C-4 position has been expected. Therefore, an object of the present invention is to provide an enzyme which transfers a sulfate group to the hydroxyl group at the C-4 position of galactosamine residue contained in galactosaminoglycan.
With a view to solving the above-mentioned problem, the present inventors have made intensive research and as a result they have now found that galactosaminoglycan 4-sulfotransferase, which transfers a sulfate group to the hydroxyl group at the C-4 position of galactosamine residue contained in galactosaminoglycan, exists in mouse chondrosarcoma cells and further they have been successful in isolating and purifying it, thereby completing the present invention.
That is, the present invention is to provide an isolated galactosaminoglycan 4-sulfotransferase which has an activity of transferring a sulfate group from a sulfate group donor to the hydroxyl group at the C-4 position of galactosamine residue contained in galactosaminoglycan (hereinafter, also referred to as “enzyme of the present invention” or “G4ST”).
The enzyme of the present invention preferably acts on chondroitin, chondroitin sulfate A, chondroitin sulfate C, or desulfated dermatan sulfate.
The enzyme of the present invention preferably further has the following properties:
Optimum reaction pH: pH 6.5-7.5;
Enhancement of activity: The activity of the enzyme is enhanced by prolamine or Ca
2+
; and
Inhibition of activity: The activity of the enzyme is inhibited by Co
2+
.
REFERENCES:
Sugumaran et al., Simultaneous Sulfation of Endogenous Chondroitin Sulfate and Chonfroitin-derived Oligosaccarides, (1986), J.B.C.vol. 261, pp. 12659-12664.*
Habuchi et al., Secretion of Condroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from the cultured chick embryo chondrocytes, (1991), B.B.A. vol. 1133, pp. 9-16.*
Habuchi and Miyashita, Seperartion and Characterization of Chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from chick embryo cartilage, (1982), BBA, vol. 717, pp. 414-421.*
Biochimica et Biophysica Acta,Secretion of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from cultured chick embryo chondrocytes,1133 (1991) 9-16, Osami Habuchi, Masafumi Tsuzuki, Ikuko Takeuchi, Masae Hara, Yasuko Matsui and Satoko Ashikari.
O. Habushi, et al. “Stimulation of Glycosaminoglycan from Chick Embryo Cartilage by Basic Proteins and Polyamines.”Biochemica et Biophysica Acta,616 (1980), pp. 208-217.
O. Habushi, et al. “Separation and Characterization of Chondroitin 6-Sulfotransferase and Chondroitin 4-Sulfotransferase from Chick Embryo Cartilage.”Biochimica de Biophysica Acta,717 (1982) pp. 414-421.
O. Habushi, et al. “Secretion of Chondroitin 6-Sulfotransferase and Chondroitin 4-Sulfotransferase from Cultured Chick Embryo Chondrocytes,”Biochimica et Biophysica Acta,1133 (1991) pp. 9-16.
H. Kitagawa, et al. “Developmental Regulation of the Sulfation Profile of Chondroitin Sulfate Chains in the Chicken Embryo Brain.”Journal of Biological Chemistry,vol. 272, No. 50, Dec., 1997. pp. 31377-31381.
S. Yamauchi, et al. “Purification and Characterization of Chondroitin 4-Sulfotransferase from the Culture Medium of a Rat Chondrosarcoma Cell Line.”The of Biological Chemistry,vol. 274, No. 4, Jan., 1999, pp. 2456-2463.
S. Yamauchi, et al. “Molecular Cloning and Expression of Chondroitin 4-Sulfotransferase.”Journal of Biological Chemistry,vol. 275, No. 12, Mar., 2000. pp. 8975-8981.
Habuchi Osami
Hirahara Yukie
Yamauchi Shinobu
Guttman Harry J
Knobbe Martens Olson & Bear LLP
Seikagaku Corporation
Weber Jon P.
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