Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-12-23
2002-09-24
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S194000, C435S183000, C530S350000
Reexamination Certificate
active
06455289
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a polypeptide of N-acetylglucosamine-6-O-sulfotransferase and a DNA having a nucleotide sequence encoding the polypeptide.
DESCRIPTION OF THE PRIOR ART
The closest prior art is described below.
It has been described in Biochem. J., 319, 209-216 (1996) and J. Biol. Chem. 272, 29493-29501 (1997) that rat and human microsome fractions had an N-acetylglucosamine-6-O-sulfotransferase activity. However, there has been so far no report about isolation and identification of a polypeptide of N-acetylglucosamine-6-O-sulfotransferase. A DNA encoding this polypeptide has not also been known.
Once a polypeptide of N-acetylglucosamine-6-O-sulfotransferase is obtained, it can be used for synthesis of sugar chains such as GlyCAM-1 that is a ligand of L-selectin (which is involved in homing of lymphocytes and rolling of leukocytes occurring at the early stage of inflammation). A DNA encoding this polypeptide would be expected to be used for large scale production of the polypeptide, or artificial synthesis of GlyCAM-1 (having a structure of NeuAc&agr;2-3Gal&bgr;1-4(Fuc&agr;1-3) (SO
4
-6)GlcNAc-) by using transformants which harbors the DNA.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a polypeptide of N-acetylglucosamine-6-O-sulfotransferase and a DNA encoding the polypeptide.
The present inventors have succeeded in cloning a DNA encoding a polypeptide of N-acetylglucosamine-6-O-sulfotransferase, having an activity to specifically transfer a sulfate group to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of GlcNAc&bgr;1-3Gal&bgr;1-4GlcNAc, wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, &bgr;1-3 represents a &bgr;1-3 glycosidic linkage, and &bgr;1-4 represents a &bgr;1-4 glycosidic linkage. The inventors have also confirmed that the polypeptide of N-acetylglucosamine-6-O-sulfotransferase was expressed by the DNA and identified the polypeptide, thereby completing the present invention.
The present invention provides a polypeptide of (a) or (b) below (hereinafter sometimes referred to as “the polypeptide of the present invention”):
(a) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2; or
(b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a sulfate group from a sulfate group donor to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of an oligosaccharide represented by the formula I:
GlcNAc&bgr;1-3Gal&bgr;1-4GlcNAc (I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, &bgr;1-3 represents a &bgr;1-3 glycosidic linkage, and &bgr;1-4 represents a &bgr;1-4 glycosidic linkage.
The polypeptide of the present invention also includes a polypeptide of (a) or (b) below:
(a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4; or
(b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a sulfate group from a sulfate group donor to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of an oligosaccharide represented by the formula I:
GlcNAc&bgr;1-3Gal&bgr;1-4GlcNAc (I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, &bgr;1-3 represents a &bgr;1-3 glycosidic linkage, and &bgr;1-4 represents a &bgr;1-4 glycosidic linkage.
The polypeptide of the present invention also includes a polypeptide having the following properties:
(1) Action: a sulfate group is transferred from a sulfate group donor to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of an oligosaccharide represented by the formula I:
GlcNAc&bgr;1-3Gal&bgr;1-4GlcNAc (I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, &bgr;1-3 represents a &bgr;1-3 glycosidic linkage, and &bgr;1-4 represents a &bgr;1-4 glycosidic linkage;
(2) Substrate specificity: sulfate group is not transferred to chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, desulfated keratan sulfate, CDSNS-heparin, mucin from porcine stomach, mucin from bovine submaxillary gland, and an oligosaccharide represented by the formula II:
Gal&bgr;1-4GlcNAc&bgr;1-3Gal&bgr;1-4GlcNAc (II)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, &bgr;1-3 represents a &bgr;1-3 glycosidic linkage, and &bgr;1-4 represents a &bgr;1-4 glycosidic linkage; and
(3) N-terminal amino acid sequence comprises an amino acid sequence represented by amino acid numbers 1 to 48 in SEQ ID NO: 2.
The present invention provides a DNA encoding a polypeptide of (a) or (b) below (hereinafter sometimes referred to as “the DNA of the present invention”):
(a) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2; or
(b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a sulfate group from a sulfate group donor to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of an oligosaccharide represented by the formula I:
GlcNAc&bgr;1-3Gal&bgr;1-4GlcNAc (I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, &bgr;1-3 represents a &bgr;1-3 glycosidic linkage, and &bgr;1-4 represents a &bgr;1-4 glycosidic linkage.
The DNA of the present invention also includes a DNA encoding a polypeptide of (a) or (b) below:
(a) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 4; or
(b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a sulfate group from a sulfate group donor to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of an oligosaccharide represented by the formula I:
GlcNAc&bgr;1-3Gal&bgr;1-4GlcNAc (I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, &bgr;1-3 represents a &bgr;1-3 glycosidic linkage, and &bgr;1-4 represents a &bgr;1-4 glycosidic linkage.
The DNA of the present invention is preferably a DNA comprising a nucleotide sequence represented by nucleotide numbers 470 to 1918 in SEQ ID NO: 1 and a DNA comprising a nucleotide sequence represented by nucleotide numbers 390 to 1841 in SEQ ID NO: 3.
The present invention also provides a method of producing a sulfated sugar (hereinafter sometimes referred to as “the producing method 1 of the present invention”) represented by the formula III:
(SO
4
-6)GlcNAc-R (III)
wherein GlcNAc represents an N-acetylglucosamine residue; SO
4
-6 means that a hydroxyl group at 6 position is sulfated, and -R represents a hydrogen atom or a sugar residue bonded by a glycosidic linkage,
which comprises a step of reacting the above-described polypeptide with a sugar chain represented by the formula IV:
GlcNAc-R (IV)
wherein GlcNAc represents an N-acetylglucosamine residue, and -R represents a hydrogen atom or a sugar residue bonded by a glycosidic linkage.
Furthermore, the present invention provides a method of producing a sulfated sugar (hereinafter sometimes referred to as “the producing method 2 of the present invention”), which comprises a step of transfecting a cell with the DNA of the present invention into and then culturing
Habuchi Osami
Kadomatsu Kenji
Kannagi Reiji
Muramatsu Hideki
Muramatsu Takashi
Hutson Richard
Knobbe Martens Olson & Bear LLP
Prouty Rebecca E.
Seikagaku Corporation
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