JP2003164291A - New chondroitin synthetase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DNA encoding a human chondroitin synthetase and its use. <P>SOLUTION: The invention relates to a protein containing an amino acid sequence corresponding to the amino acid number 42 to 532 of a specific sequence, a substance having the effect same as that of the protein and a DNA encoding the protein, etc. A new human-derived chondroitin synthetase can be produced by the expression of the DNA. A sugar chain containing GalNAc including chondroitin can be produced by using the human chondroitin synthetase. The DNA or a part of the DNA can be used as a probe for the hybridization of the human-derived chondroitin synthetase. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel chondroitin synthase, a DNA encoding the same, a vector containing this DA, a transformant transformed with this vector, a method for producing the chondroitin synthase, GalN.
The present invention relates to a method for producing a sugar chain containing Ac or GlcNAc, and a hybridization probe for the chondroitin synthase.

[0002]

2. Description of the Related Art First, symbols and abbreviations commonly used in this specification are as follows. GalNAc: N-acetyl-D-galactosamine GlcUA: D-glucuronic acid GlcNAc: N-acetyl-D-glucosamine Gal: galactose Xyl: xylose UDP: uridine 5'-diphosphate Cbz: benzyloxycarbonyl Ser: serine GAG (or GAGs): Glycosaminoglycan GalNAcT: Chondroitin β1,4-N-acetylgalactosaminyltransferase GlcAT: β1,3-Glucuronyltransferase HPLC: High performance liquid chromatography MES: 2- (N-morpholino) ethanesulfonic acid

At least one GAG in the core protein
Chondroitin / dermatan sulphate proteoglycans with covalently linked chains are a widespread constituent of the extracellular matrix of connective tissues and are also present on the cell surface and intracellular secretory granules of many cell types. The GAG chain shows tissue-specific and developmentally regulated expression, and is also involved in cell proliferation, cell differentiation, and regulation and maintenance of tissue morphology (References 1 to 3). In particular, recent studies on chondroitin / dermatan sulfate chains have been shown to play an important role in the formation of neural networks in the developing mammalian brain, and thus have been noted (References 4 and 5).

Chondroitin / dermatan sulfate, like heparin / heparan sulfate, is a so-called GAG-protein linkage site (GlcUAβ1-3Galβ1-3Galβ1-4Xylβ1-O-; bound to the Ser residue of the core protein; chondroitin / dermatan sulfate and heparin). / Common to GAGs containing heparan sulfate) (6 and 7). The synthesis of the linkage site begins with the addition of Xyl to Ser, followed by the addition of two Gal residues, and is completed by the addition of GlcUA, each reaction catalyzed by a specific glycosyltransferase. (References 6 and 7). GA
Gs is constructed by alternating N-acetylhexosamine and GlcUA at this linkage site. Chondroitin / dermatan sulfate is synthesized by first transferring GalNAc to the common linkage site, and heparin / heparan sulfate is synthesized by first transferring GlcNAc. Thus, the initial hexosamine transfer is believed to be a critical step for the selective assembly of chondroitin / dermatan sulfate and heparin / heparan sulfate chains at a common linkage site. However, this biosynthetic mechanism has been proposed for a long time based on conventional structural and enzymatic studies (Reference 7), and the glycosyltransferase that plays this role has not been molecularly cloned. It was In particular, the molecular mechanism underlying the selective chain biosynthesis of different types of GAG chains has been elucidated by cDNA cloning of glycosyltransferases, which are recently related to heparin / heparan sulfate (Reference 8), but are long. It was still a mystery.

CDNA cloning of a glycosyltransferase involved in heparin / heparan sulfate biosynthesis has shown that the EXT gene family is involved in its biosynthesis (Reference 8). Both human EXT1 and EXT2 proteins have GlcUA and GlcNAc
It has been shown to be a heparan sulfate co-polymerase that alternately polymerizes heparin sulfate (References 9 to 11), and human EXTL1 protein is GlcNAc transferase II involved in the extension of heparan sulfate chain (Reference 12), and human EXTL2 protein. Gl determines and initiates heparan sulfate synthesis at a common GAG-protein linkage site
cNAc transferase I (Reference 13), human E
The XTL3 protein is GlcNAc transferase I and I
It has been shown to have both activities of I (Reference 1).
2).

On the other hand, the mechanism of the construction of chondroitin sulfate chains has not been completely clarified. We have recently demonstrated that GlcUA transferase II (GlcAT-II) and GalNA are responsible for the biosynthesis of repeating disaccharide units of chondroitin sulfate.
A cDNA encoding a chondroitin synthase having a c-transferase II (GalNAcT-II) activity (hereinafter, this enzyme is referred to as "chondroitin synthase 1") was cloned (Reference 15). A single protein with two glycosyltransferase activities is reminiscent of the heparan sulfate co-enzyme encoded by EXT1 and EXT2. However, by analyzing the receptor substrate specificity of recombinant human chondroitin synthase, this enzyme is not GalNAc transferase I (GalNAcT-I) that determines and initiates chondroitin sulfate synthesis at a common GAG-protein linkage site (Reference 16). ), It was considered that there is at least one other gene encoding GalNAcT-I in the human genome.

[0007]

The present invention provides a novel chondroitin synthase that triggers biosynthesis of chondroitin, a vector containing a DNA encoding the same, a transformant transformed with this vector, and the chondroitin synthesis. An object of the present invention is to provide a method for producing an enzyme, a method for producing a sugar chain containing GalNAc or GlcNAc, and a hybridization probe for the chondroitin synthase.

[0008]

Means for Solving the Problems The present inventors have conducted an intensive search of a database to selectively biosynthesis (ie, initiation and extension) of chondroitin / dermatan sulfate chains.
We have succeeded in finding a candidate for a DNA encoding GalNAc transferase (a novel chondroitin synthase derived from human), which is a key enzyme responsible for the enzyme. And candidate D
By actually expressing NA, it was confirmed that the candidate DNA was a DNA encoding chondroitin synthase that triggers chondroitin biosynthesis, and the present invention was completed.

That is, the present invention provides the following: (1) The following protein (A) or (B) (hereinafter, "the protein of the present invention" or "chondroitin synthase 2")
Say. ). (A) A protein containing the amino acid sequence represented by amino acid numbers 42 to 532 in SEQ ID NO: 2. (B) A protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or transposed in the amino acid sequence of (A) above and having the following properties (a) and (b). (A) It has a catalytic activity for transferring GalNAc from UDP-GalNAc to chondroitin. (UDP represents uridine 5'-diphosphate and GalNAc represents an N-acetylgalactosamine residue.) (B) It has substantially no catalytic activity for transferring GlcUA from UDP-GlcUA to chondroitin. (UDP is Uridine 5'-
For diphosphate, GlcUA indicates a glucuronic acid residue. (2) In addition to the above (a) and (b), the following (c)
And (2) the protein described in (1). (C) From α-thrombomodulin to UDP-GalNAc to GalN
It has catalytic activity to transfer Ac. (UDP represents uridine 5'-diphosphate, GalNAc represents an N-acetylgalactosamine residue.) (D) GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz has a catalytic activity of transferring GalNAc from UDP-GalNAc. . (UDP represents uridine 5'-diphosphate, GlcUA represents a glucuronic acid residue, Gal represents a galactose residue, GalNAc represents an N-acetylgalactosamine residue, and Cbz represents a benzyloxycarbonyl group.) (3) The protein according to (1) or (2), wherein the protein (A) is a protein having an amino acid sequence represented by amino acid numbers 42 to 532 in SEQ ID NO: 2. (4) In (1) or (2), the protein of (A) is a fusion protein of a protein consisting of the amino acid sequence represented by amino acid numbers 42 to 532 in SEQ ID NO: 2 and another peptide or polypeptide. The described protein. (5) The protein according to (1) or (2), wherein the protein (A) is a protein having an amino acid sequence represented by amino acid numbers 1 to 532 in SEQ ID NO: 2. (6) The protein is a soluble protein, (1)
~ The protein according to any one of (5). (7) A vector carrying the DNA of any of the following (a) to (c) (hereinafter referred to as the vector of the present invention). (A) D encoding a protein containing the amino acid sequence represented by amino acid numbers 42 to 532 in SEQ ID NO: 2
NA. (B) The amino acid sequence represented by amino acid numbers 42 to 532 in SEQ ID NO: 2 contains an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or transposed, and has the following properties (a) and (b): DNA encoding a protein having: (C) hybridizes with the DNA described in (a) above, a DNA complementary to the DNA, or a DNA having a part of the base sequence of these DNAs under stringent conditions, and (a) and A DNA encoding a protein having the property (b). (A) It has a catalytic activity for transferring GalNAc from UDP-GalNAc to chondroitin. (UDP represents uridine 5'-diphosphate and GalNAc represents an N-acetylgalactosamine residue.) (B) It has substantially no catalytic activity for transferring GlcUA from UDP-GlcUA to chondroitin. (UDP is Uridine 5'-
For diphosphate, GlcUA indicates a glucuronic acid residue. (8) In addition to the above (a) and (b), the following (c)
And a DN encoding a protein having (d) properties
The vector according to (7), which retains A. (C) From α-thrombomodulin to UDP-GalNAc to GalN
It has catalytic activity to transfer Ac. (UDP is Uridine 5'-
Diphosphate and GalNAc represent N-acetylgalactosamine residues. (D) It has catalytic activity for transferring GalNAc from UDP-GalNAc to GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz. (UDP is uridine 5'-diphosphate, GlcUA is a glucuronic acid residue, Gal is a galactose residue, GalNAc is an N-acetylgalactosamine residue, and Cbz is a benzyloxycarbonyl group.) (9) The DNA of (a) above has amino acid numbers 42 to 53.
The vector according to (7) or (8), which is a DNA encoding a protein consisting of the amino acid sequence shown in 2. (10) The vector according to (9), wherein the DNA encoding the protein consisting of the amino acid sequence represented by amino acid numbers 42 to 532 is the DNA represented by nucleotide numbers 778 to 2250 in SEQ ID NO: 1. (11) The DNA of (a) above has amino acid numbers 42 to 5
The vector according to (7) or (8), which is a DNA encoding a fusion protein of a protein consisting of the amino acid sequence represented by 32 and another peptide or polypeptide. (12) The DNA encoding the fusion protein of the protein consisting of the amino acid sequence represented by amino acid numbers 42 to 532 and another peptide or polypeptide is the DNA represented by nucleotide numbers 778 to 2250 in SEQ ID NO: 1 and other peptides. Alternatively, the vector according to (11), which is a ligation product with a DNA encoding a polypeptide. (13) The DNA of (a) above has amino acid numbers 1 to 53.
The vector according to (7) or (8), which is a DNA encoding a protein consisting of the amino acid sequence shown in 2. (14) The vector according to (13), wherein the DNA encoding the protein consisting of the amino acid sequence represented by amino acid numbers 1 to 532 is the DNA represented by nucleotide numbers 655 to 2250 in SEQ ID NO: 1. (15) The protein is a soluble protein,
The vector according to any one of (7) to (14). (16) The vector according to any one of (7) to (15), which is an expression vector. (17) A transformant obtained by transforming a host with the vector according to any one of (7) to (16) (hereinafter,
The transformant of the present invention). (18) A method for producing chondroitin synthase, which comprises growing the transformant according to (17) and collecting chondroitin synthase from the grown product (hereinafter, referred to as
The enzyme production method of the present invention). (19) A reagent for synthesizing an N-acetylgalactosamine-containing sugar chain, which contains the protein according to any one of (1) to (6) (hereinafter referred to as the reagent of the present invention). (20) A method for producing a sugar chain represented by the following general formula (2), which comprises at least a step of contacting the reagent described in (19) with a GalNAc donor and a sugar chain represented by the following general formula (1) ( Hereinafter, the sugar chain production method 1 of the present invention). ^{GlcUA-GalNAc-R 1 (1} ) GalNAc-GlcUA-GalNAc-R 1 (2) ( in each formula, GlcUA and GalNAc are both the same meaning as defined above -. The glycosidic bond, -R ¹ is optional (21) A sugar represented by the following general formula (4), which comprises at least a step of bringing the reagent described in (21) or (19) into contact with a GalNAc donor and a sugar chain represented by the following general formula (3). Chain production method (hereinafter referred to as the sugar chain production method 2 of the present invention. Further, the sugar chain production methods 1 and 2 of the present invention will be collectively referred to as the “method of the present invention sugar chain”
That). GlcUA-Gal-R ¹ (3) GalNAc-GlcUA-Gal-R ¹ (4) (In each formula, GlcUA, GalNAc and Gal are as defined above.- is a glycosidic bond and -R ¹ is (Indicates any group.) (22) Nucleotide number 655 to SEQ ID NO: 1
A hybridization probe having a sequence complementary to the nucleotide sequence represented by 2250 or a part thereof (hereinafter, referred to as
The present invention probe). (23) The following protein (A) or (B) (hereinafter,
Also referred to as "chondroitin synthase 3"). (A) A protein comprising the amino acid sequence represented by amino acid numbers 58 to 772 in SEQ ID NO: 6. (B) A protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or transposed in the amino acid sequence of (A) above and having the following properties (a) and (b). (A) UDP-GlcU is added to the chondroitin heptasaccharide shown below.
It has catalytic activity to transfer GlcUA from A. GalNAc- (GlcUAβ1-3GalNAc) ₃ (UDP indicates uridine 5′-diphosphate, GlcUA indicates glucuronic acid residue, GalNAc indicates N-acetylgalactosamine residue.) (B) From chondroitin to UDP-GalNAc It has substantially no catalytic activity to transfer GalNAc. (UDP is Uridine 5 '
-Diphosphate, GalNAc represents an N-acetylgalactosamine residue. (24) The protein according to (23), wherein the protein of (A) is a protein having an amino acid sequence represented by amino acid numbers 58 to 772 in SEQ ID NO: 6. (25) The protein according to (23), wherein the protein (A) is a fusion protein of a protein consisting of the amino acid sequence represented by amino acid numbers 58 to 772 in SEQ ID NO: 6 and another peptide or polypeptide. (26) The protein according to (23), wherein the protein (A) is a protein having an amino acid sequence represented by amino acid numbers 1 to 772 in SEQ ID NO: 6. (27) The protein is a soluble protein, (2
The protein according to any one of 3) to (26). (28) A vector carrying the DNA according to any one of the following (a) to (c). (A) D encoding a protein containing the amino acid sequence represented by amino acid numbers 58 to 772 in SEQ ID NO: 6
NA. (B) the amino acid sequence represented by amino acid numbers 58 to 772 in SEQ ID NO: 2 contains an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or transposed, and has the following properties (a) and (b) DNA encoding a protein having: (C) hybridizes with the DNA described in (a) above, a DNA complementary to the DNA, or a DNA having a part of the base sequence of these DNAs under stringent conditions, and (a) and A DNA encoding a protein having the property (b). (A) UDP-GlcU is added to the chondroitin heptasaccharide shown below.
It has catalytic activity to transfer GlcUA from A. GalNAc- (GlcUAβ1-3GalNAc) ₃ (UDP indicates uridine 5′-diphosphate, GlcUA indicates glucuronic acid residue, GalNAc indicates N-acetylgalactosamine residue.) (B) From chondroitin to UDP-GalNAc It has substantially no catalytic activity to transfer GalNAc. (UDP is Uridine 5 '
-Diphosphate, GalNAc represents an N-acetylgalactosamine residue. (29) The DNA of (a) above has amino acid numbers 58 to 7
The vector according to (28), which is a DNA encoding a protein consisting of the amino acid sequence represented by 72. (30) The vector according to (29), wherein the DNA encoding the protein consisting of the amino acid sequence represented by amino acid numbers 58 to 772 is the DNA represented by nucleotide numbers 1685 to 3829 in SEQ ID NO: 5. (31) The DNA of (a) above has amino acid numbers 58 to 7
The vector according to (28), which is a DNA encoding a fusion protein of the protein consisting of the amino acid sequence represented by 72 and another peptide or polypeptide. (32) The DNA encoding the fusion protein of the protein consisting of the amino acid sequence represented by amino acid numbers 58 to 772 and another peptide or polypeptide is the DNA represented by nucleotide numbers 1685 to 3829 in SEQ ID NO: 1 and other peptides. Alternatively, the vector according to (31), which is a ligation product with a DNA encoding a polypeptide. (33) The DNA of (a) above has amino acid numbers 1 to 77.
The vector according to (28), which is a DNA encoding a protein consisting of the amino acid sequence shown in 2. (34) The vector according to (33), wherein the DNA encoding the protein consisting of the amino acid sequence represented by amino acid numbers 1 to 772 is the DNA represented by nucleotide numbers 1514 to 3829 in SEQ ID NO: 5. (35) the protein is a soluble protein,
The vector according to any one of (28) to (34). (36) The vector according to any one of (28) to (35), which is an expression vector. (37) A transformant obtained by transforming a host with the vector according to any one of (28) to (36). (38) A method for producing chondroitin synthase, which comprises growing the transformant according to (37) and collecting chondroitin synthase from the grown product. (39) A glucuronic acid-containing sugar chain synthesizing reagent containing the protein according to any one of (23) to (27). (40) A method for producing a sugar chain represented by the following general formula (6), which comprises at least a step of contacting the reagent described in (39) with a GlcUA donor and a sugar chain represented by the following general formula (5). GalNAc-R ¹ (5) GlcUA-GalNAc-R ¹ (6) (In each formula, GlcUA and GalNAc have the same meanings as described above.-Represents a glycosidic bond and -R ¹ represents an arbitrary group. (41) Nucleotide number 1514 in SEQ ID NO: 5
A probe for hybridization having a base sequence represented by 3829 or a sequence complementary to a part thereof.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION <1> Protein of the Present Invention The protein of the present invention is the following protein (A) or (B). (A) A protein containing the amino acid sequence represented by amino acid numbers 42 to 532 in SEQ ID NO: 2. (B) A protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or transposed in the amino acid sequence of (A) above and having the following properties (a) and (b). (A) It has a catalytic activity for transferring GalNAc from UDP-GalNAc to chondroitin. (B) It has substantially no catalytic activity for transferring GlcUA from UDP-GlcUA to chondroitin.

Here, chondroitin refers to GlcUA and GalNAc.
A polymer composed of repeating disaccharide units of ## STR3 ## which contains both the non-reducing end of which is GlcUA and the one of which is GalNAc. And, the transfer of GalNAc is such that the non-reducing end is Gl.
It can be said that it is against cUA, chondroitin.

SEQ ID NO: 2 as described in the Examples below.
It has been confirmed that the protein containing the amino acid sequence represented by amino acid Nos. 42 to 532 of the amino acid sequence shown in 1) has the enzymatic activity of chondroitin synthase. Amino acid numbers 1 to 4 of the amino acid sequence shown in SEQ ID NO: 2
The part of the amino acid sequence shown by 1 is considered to include a transmembrane region. Therefore, a soluble protein of the present invention (chondroitin synthase) can be obtained by using a protein that does not contain the amino acid sequence represented by amino acid numbers 1 to 41. Soluble protein is a protein that normally does not have a transmembrane domain, and means a protein that is soluble in an aqueous solvent such as water or a buffer when expressed. Soluble proteins are preferred because they are easily purified.

The protein of the present invention is preferably such a soluble protein. Among them, a protein having an amino acid sequence represented by amino acid numbers 42 to 532 is preferable.

A protein containing the amino acid sequence represented by amino acid Nos. 42 to 532 of the amino acid sequence shown in SEQ ID No. 2 is:
It may be a fusion protein with a peptide such as a signal peptide or a polypeptide having another function. For example, a fusion protein with a marker peptide is particularly preferable when purifying the protein of the present invention. Examples of such marker peptides include protein A, insulin signal sequence, His, FLAG, CBP (calmodulin binding protein), GST (glutathione S-transferase) and the like. For example, if it is fused with protein A, affinity purification can be easily performed, and if it is fused with an insulin signal sequence or the like, the enzyme can be secreted extracellularly (medium etc.). In addition, a fusion protein with a polypeptide having another function is also preferable in that it can be a protein having both the function of the protein of the present invention as a chondroitin synthase and the function of another polypeptide. . Examples of other polypeptides include, but are not limited to, chondroitin synthase 1, chondroitin synthase 3, sulfate group transferase (sulfotransferase), and the like. When a fusion protein with uronic acid epimerase (C5 epimerase) is used, it can be applied to the production of dermatan sulfate.

As described above, the protein (A) is
It is preferably a fusion protein of a protein having the amino acid sequence represented by amino acid numbers 42 to 532 and another peptide or polypeptide.

Of course, the protein of the present invention may be a protein containing an amino acid sequence represented by amino acid Nos. 1 to 41, if necessary. That is, as the protein (A), a protein having an amino acid sequence represented by amino acid numbers 1 to 532 may be adopted.

In addition, naturally occurring proteins include polymorphisms and mutations of the DNA encoding them, as well as substitution of amino acids in the amino acid sequence of the produced protein due to modification reactions in the cells and during purification. It is known that some mutations such as deletion, insertion or rearrangement may occur, but nonetheless exhibit substantially the same physiological / biological activity as the protein having no mutation. The proteins of the present invention also include proteins in which there is no significant difference in their functions even if there are slight structural differences. The same is true when artificially introducing the above-mentioned mutation into the amino acid sequence of the protein, and in this case, it is possible to prepare a wider variety of mutants.

Further, in order to cause substitution, deletion, insertion or transposition of amino acids in the amino acid sequence, it is possible to artificially substitute, delete, insert or transpose the bases in the DNA base sequence.

Such substitution, deletion, insertion or rearrangement of the bases in the base sequence of DNA synthesizes, for example, a sequence containing a mutation point and having restriction enzyme cleavage ends at both ends,
It can be introduced by replacing the corresponding portion of the base sequence of unmutated DNA. In addition, site-directed mutagenesis (Kremer, W. and Frits, HJ, Meth.
Enzymol., 154, 350 (1987); Kunkel, TA et al., Me
th. In Enzymol., 154, 367 (1987)) and the like can also introduce base substitution, deletion, insertion or rearrangement into DNA.

It is known that certain proteins have a peptide region which is not essential for activity. This includes, for example, signal peptides present in extracellularly secreted proteins and prosequences found in protease precursors, etc., and most of these regions are removed post-translationally or upon conversion to an active protein. It Such proteins exist in different forms in terms of their primary structure, but ultimately have equivalent functions. Examples of such proteins include the proteins of (B) above.

[0021] In the present specification, "several amino acids"
Means the number of amino acids that may be mutated to the extent that the catalytic activity of (a) and (b) described below is not lost. For example, in the case of a protein consisting of about 600 amino acid residues, about 3 to 30 The number is preferably 3 to 15, and more preferably 3 to 8.

The above catalytic activities (a) and (b) can be detected by a general assay method for glycosyltransferase. Specifically, as shown in Examples below, it can be measured by a measurement method using UDP-GalNAc as a donor and a transfer reaction of GalNAc to chondroitin. Therefore, those skilled in the art can use the presence or absence of these transposition activities as an index and do not substantially impair the activity.
Substitution of one or more, especially one or several amino acid residues,
Deletions, insertions or transpositions can be easily selected.

The chondroitin used here includes:
The non-reducing end includes both those having GlcUA and GalNAc, and the chondroitin to which GalNAc is transferred by the action of the protein of the present invention is a chondroitin having a non-reducing end being GlcUA. As I did.

The protein (B) preferably has the following properties (c) and (d) in addition to the properties (a) and (b): (C) From α-thrombomodulin to UDP-GalNAc to GalN
It has catalytic activity to transfer Ac. The α-thrombomodulin contains a tetrasaccharide consisting of GlcUAβ1-3Galβ1-3Galβ1-4Xyl. (D) It has a catalytic activity for transferring GalNAc from UDP-GalNAc to GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz.

The catalytic activities of (c) and (d) above can also be detected by a general assay method for glycosyltransferase as described above. See the examples for details.

Since the amino acid sequence, properties, etc. of the protein of the present invention have been disclosed in the present specification, it can be produced by various methods such as genetic engineering techniques and chemical synthesis methods based on the disclosure. Among them, the production method of the present invention described later is simple and preferable.

<2> Vector of the Present Invention The vector of the present invention contains D of any one of the following (a) to (c):
It is a vector carrying NA. (A) D encoding a protein containing the amino acid sequence represented by amino acid numbers 42 to 532 in SEQ ID NO: 2
NA. (B) The amino acid sequence represented by amino acid numbers 42 to 532 in SEQ ID NO: 2 contains an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or transposed, and has the following properties (a) and (b): DNA encoding a protein having: (C) hybridizes with the DNA described in (a) above, a DNA complementary to the DNA, or a DNA having a part of the base sequence of these DNAs under stringent conditions, and (a) and A DNA encoding a protein having the property (b). (A) It has a catalytic activity for transferring GalNAc from UDP-GalNAc to chondroitin. (B) It has substantially no catalytic activity for transferring GlcUA from UDP-GlcUA to chondroitin.

Those skilled in the art are aware that, as the DNAs (a) to (c) carried by the vector of the present invention, there are DNAs having various different nucleotide sequences due to the degeneracy of the genetic code. It is easy to understand.

The description of the protein encoded by the DNA of (a) above is the same as the description of the protein of (A) in the above-mentioned "<1> Protein of the present invention".

The DNAs of (b) and (c) above are
It is intended to include a DNA encoding a protein that has a different primary structure from the protein encoded by the DNA of (a) but has a similar function. The description of the protein encoded by the DNAs of (b) and (c) is the same as the description of the protein of (B) in the above-mentioned “<1> protein of the present invention”.

The "stringent conditions" in (c) above means conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed (Sambrook, J. et al., Molecular Cloning). A Lab
oratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press (1989) etc.). Specifically, as “stringent conditions”, 50% formamide, 4 ×
SSC, 50 mM HEPES (pH 7.0), 10 x Denhardt's so
Solution, hybridize at 42 ℃ in a solution containing 100 μg / ml salmon sperm DNA, then 2 × SSC, 0.1% S at room temperature.
The conditions for washing with a DS solution and a 0.1 × SSC, 0.1% SDS solution at 50 ° C. may be mentioned.

For example, among the DNAs that hybridize under such stringent conditions, DN encoding the protein having the catalytic activity of (a) and (b) above
A can be easily selected. The methods for detecting the catalytic activity in (a) and (b) above are the same as those described in "<1> Protein of the present invention".

The proteins encoded by the DNAs of (b) and (c) above are the same as (a) and (b) above.
In addition to the above, a protein having the following properties (c) and (d) is preferable. (C) From α-thrombomodulin to UDP-GalNAc to GalN
It has catalytic activity to transfer Ac. (D) It has a catalytic activity for transferring GalNAc from UDP-GalNAc to GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz.

The method of detecting the catalytic activity of (c) and (d) above is the same as described in "<1> Protein of the present invention". Further, as described in “<1> Protein of the present invention”, the protein of the present invention is preferably a soluble protein. Therefore, the protein encoded by the DNA of the above (a), (b) and (c) The invention protein) is also preferably a soluble protein. Specifically, the DNA of (a) is preferably a DNA encoding a protein consisting of the amino acid sequence represented by amino acid numbers 42 to 532, and the DNA represented by nucleotide numbers 778 to 2250 in SEQ ID NO: 1. More preferably.

Further, as described in "<1> Protein of the present invention", the protein of the present invention has amino acid numbers from 42 to 42.
A protein consisting of the amino acid sequence represented by 532.
Since it may be a fusion protein with a peptide such as a marker peptide or a signal peptide, or a polypeptide having another function, the protein encoded by the DNA of the above (a), (b) and (c) (the present The invention protein) may be such a fusion protein. Specifically, the DNA (a) is preferably a DNA encoding a fusion protein of a protein consisting of the amino acid sequence represented by amino acid numbers 42 to 532 and another peptide or polypeptide, and SEQ ID NO: 1 It is preferably a ligated product of the DNA represented by nucleotide numbers 778 to 2250 in 1 above and the DNA encoding another peptide or polypeptide. The description of “other peptide or polypeptide” that can be fused is the same as the description of “<1> protein of the present invention”.

Further, as described in "<1> Protein of the present invention", the protein of the present invention may be a protein containing the amino acid sequence represented by amino acid Nos. 1 to 41, if necessary. The protein encoded by DNA may be such a protein. Specifically, the DNA of (a) above is preferably a DNA encoding a protein having an amino acid sequence represented by amino acid numbers 1 to 532, and is a DNA represented by nucleotide numbers 655 to 2250 in SEQ ID NO: 1. More preferably.

Since the vector of the present invention is preferably used in the method for producing chondroitin synthase described later, it is preferably an expression vector.

For example, DNA encoding the amino acid sequence shown by amino acid Nos. 42 to 532 (amino acid No. 1
An expression vector carrying a sequence (not including the sequence encoding the amino acid sequence represented by ~ 41) can be prepared by the following method.

<A> Preparation of DNA to be incorporated in vector Using total RNA of human-derived cells (for example, G361 human melanoma cells (ATCC CRL-1424)) as a template, 5'-primer containing a BamHI site (5'-CGGGATCCCAGCTG
GCACTGCCCAGG-3 ') (SEQ ID NO: 3) and 58 bp of stop codon
3'-primer (5'-CG containing BamHI site located downstream
Reverse transcription-PCR using GGATCCCATCTCTGACCCATCAGTCC-3 ') (SEQ ID NO: 4).

A general method can be used for the PCR reaction. For example, Pf in 5% (v / v) dimethylsulfoxide can be used.
u polymerase (Stratagene, La Jolla, CA) at 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C.
It can be carried out by repeating the 180-second cycle for 30 cycles.

<B> Introduction of DNA Fragment into Vector The vector of the present invention can be produced by introducing the DNA obtained by the above method into a known vector.

As the vector for introducing the above-mentioned DNA, for example, an appropriate expression vector (preferably containing a regulatory sequence such as a promoter) capable of expressing the introduced DNA (phage vector or plasmid vector etc.) is used. A vector capable of expressing the above DNA in a host cell into which the vector of the present invention is incorporated is appropriately selected. Such a host-vector system includes
Mammalian cells such as COS cells and 3LL-HK46 cells, and pGIR201
(Kitagawa, H., and Paulson, JC (1994) J. Biol.
Chem. 269, 1394-1401), pEF-BOS (Mizushima, S., a
nd Nagata, S. (1990) Nucleic Acid Res. 18, 532
2), pCXN2 (Niwa, H., Yamanura, K. and Miyazaki, J.
(1991) Gene 108, 193-200), pCMV-2 (Eastman
Kodak (Eastman Kodak)), pCEV18, pME18S (Maruyama et al., Med. Immunol., 20, 27 (1990)) or a combination of expression vectors for mammalian cells such as pSVL (Pharmacia Biotech), E. coli. ), PTrcHis (Invitrogen), pGEX (Pharmacia Biotech), pTrc99 (Pharmacia Biotech),
pKK233-3 (Pharmacia Biotech), pEZZZ1
8 (Pharmacia Biotech), pCH110 (Pharmacia Biotech), pET (Stratagene), pBAD (Invitrogen), pRSET (Invitrogen), and pSE420 (Invitrogen). In addition to the combination with expression vectors for prokaryotic cells such as, for example, insect cells, yeast, Bacillus subtilis, etc. are exemplified as host cells, and various vectors corresponding thereto are exemplified. Among the above host-vector systems, the combination of mammalian cells and pEF-BOS is particularly preferable.

As the vector incorporating the above-mentioned DNA, one constructed so as to express a fusion protein of a protein encoded by the incorporated DNA and a marker peptide or a signal peptide can be used. It is particularly preferable when the expressed protein of the present invention (chondroitin synthase) is purified. Examples of the peptide include protein A, insulin signal sequence, His, FLAG, CBP (calmodulin binding protein), GST (glutathione S-transferase).
And so on. If it is fused with protein A, affinity purification can be easily performed, and if it is fused with insulin signal sequence etc., the enzyme is extracellular (medium etc.)
Can be secreted by

Regardless of which vector is used, it is treated with a restriction enzyme or the like according to a conventional method so that the above DNA and vector can be ligated, and blunting or ligation of sticky ends is carried out as necessary. After that, the DNA can be ligated to the vector.

Specifically, for example, the DNA (PCR fragment) obtained in <A> above is digested with BamHI, and this is digested with pGIR201pro.
tA (J. Biol. Chem., 269, 1394-1401 (1994)) subcloned into the BamHI site of the vector, the DNA encoded by the DNA obtained in <A> above, and the insulin signal sequence and protein in the vector Fuse with the A sequence. NheI fragment containing this fusion protein sequence
Expression vector pEF-BOS (Nucleic Acid Res., 18, 5322 (1
990)) can be inserted into the XbaI site to obtain an expression vector expressing the protein of the present invention fused with the insulin signal sequence and protein A.

<3> Transformant of the Present Invention The transformant of the present invention is a transformant obtained by transforming a host with the vector of the present invention.

The "host" referred to here may be any one that can be recombined with the vector of the present invention, but one that can exhibit the function of the DNA carried by the vector of the present invention or a recombinant vector incorporating the DNA. preferable. The host includes animal cells, plant cells, microbial cells (bacteria), COS cells (COS-1 cells, COS-7 cells, etc.), 3LL-HK46.
Examples thereof include mammalian cells such as cells, E. coli, insect cells, yeast, Bacillus subtilis, and the like. The host can be appropriately selected according to the vector of the present invention.
When using the vector of the present invention based on pEF-BOS, it is preferable to select cells of mammalian origin.
COS cells are preferred.

Transformation of the host with the vector of the present invention comprises
It can be performed by a conventional method. For example, the vector of the present invention can be introduced into a host for transformation by a method using a commercially available transfection reagent, a DEAE-dextran method, an electroporation method, a method using a gene gun, or the like.

The transformant of the present invention thus obtained can be used for the production of the protein of the present invention as described later.

<4> Method for Producing Enzyme of the Present Invention The method for producing the enzyme of the present invention comprises growing the transformant of the present invention,
It is characterized in that chondroitin synthase (protein of the present invention) is collected from the grown product.

The term "growth" is a concept including the growth of cells which are the transformants of the present invention and the microorganisms themselves, and the growth of animals, insects and the like which incorporate the cells which are the transformants of the present invention. In addition, the term "growth product" as used herein is a concept including a medium (supernatant of a culture solution) after growing the transformant of the present invention, a cultured host cell, a secreted product, an excreted product, and the like. .

Growth conditions (medium, culture conditions, etc.) are appropriately selected according to the host to be used. According to this production method, various forms of chondroitin synthase can be produced depending on the transformant used. For example, as the vector of the present invention, amino acid number 42 in SEQ ID NO: 2 is used.
A soluble chondroitin synthase is produced by growing a transformant transformed with an expression vector carrying a DNA encoding the amino acid sequence represented by ˜532.

Amino acid number 1 in SEQ ID NO: 2
The insoluble (membrane-bound) chondroitin synthase is produced by growing a transformant transformed with an expression vector carrying a DNA encoding the amino acid sequence represented by ˜532.

Furthermore, if a transformant transformed with an expression vector constructed to express a fusion protein with another peptide such as a marker peptide or a signal peptide is grown, chondroitin fused with the above-mentioned other peptide can be obtained. Synthetic enzymes are produced.

Collection of chondroitin synthase from the grown product can be carried out by a known protein extraction / purification method depending on the form of the chondroitin synthase produced.

For example, when chondroitin synthase is produced in a soluble form that is secreted into the medium (supernatant of the culture medium), the medium may be collected and used as it is as chondroitin synthase. When chondroitin synthase is produced in a soluble form that is secreted into the cytoplasm or in an insoluble (membrane-bound) form, a method using a nitrogen cavitation device, homogenization, glass bead mill method, sonication, osmotic shock The chondroitin synthase can be extracted by a treatment operation such as cell crushing such as a method or freeze-thaw method, a detergent extraction, or a combination thereof, and the extract may be directly used as the chondroitin synthase.

Chondroitin synthase can be further purified from these media and extracts, and is preferable. The purification may be incomplete purification (partial purification) or complete purification, and can be appropriately selected depending on the purpose of use of the chondroitin synthase.

Specific examples of the purification method include salting out with ammonium sulfate (ammonium sulfate) and sodium sulfate, centrifugation, dialysis, ultrafiltration, adsorption chromatography, and the like.
Ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, gel filtration method, gel permeation chromatography, affinity chromatography, electrophoresis method and the like, and treatment operations such as a combination thereof can be mentioned.

For example, when chondroitin synthase is produced as a fusion protein with protein A, IgG
It can be easily purified by affinity chromatography using a solid phase to which is bound. Similarly,
When produced as a fusion protein with His, a solid phase bound with magnetic nickel can be used, and when produced as a fusion protein with FLAG, a solid phase bound with an anti-FLAG antibody can be used. Furthermore, by fusion with an insulin signal, extraction operations such as cell disruption become unnecessary.

Production of the purified chondroitin synthase can be confirmed by analyzing the amino acid sequence, action, substrate specificity and the like.

<5> Reagent of the Present Invention The reagent of the present invention is an N-acetylgalactosamine-containing sugar chain synthesis reagent containing the protein of the present invention. The protein of the present invention is as described above in “<1> Protein of the present invention”.

The reagent of the present invention has the “transfer activity of GalNAc” possessed by the protein of the present invention (chondroitin synthase),
It is applied as a synthetic reagent for a sugar chain containing N-acetylgalactosamine.

The "N-acetylgalactosamine-containing sugar chain" includes all N-acetylgalactosamine-containing sugar chains such as chondroitin, and chondroitin is preferable.

In the present specification, “synthesis of chondroitin” or “synthesis of chondroitin” is a concept including extending and extending the sugar chain of chondroitin by transferring / adding sugar to chondroitin.

The form of the reagent of the present invention is not limited and may be any of solution form, frozen form and lyophilized form. Further, other components (eg, a reagent-acceptable carrier) may be contained as long as they do not affect the activity of chondroitin synthase. Further, the reagent of the present invention may contain one or more other enzyme proteins in addition to the protein of the present invention. Examples of such enzyme proteins include, but are not limited to, chondroitin synthase 1, chondroitin synthase 3, and sulfotransferase (sulfotransferase). In addition, epimerase of uronic acid (C5
If it contains epimerase), it can be used as a reagent for dermatan sulfate synthesis. Further, the reagent of the present invention containing no other enzyme protein may be used in combination with such other enzyme protein.

<6> Method for Producing Sugar Chain of the Present Invention All methods for producing sugar chain of the present invention use the reagent of the present invention, and the method of producing sugar chain of the present invention 1 and the method of the present invention depend on the acceptor substrate used. It is divided into the sugar chain production method 2. <6-1> Sugar chain production method 1 of the present invention The sugar chain production method 1 of the present invention comprises at least the step of bringing the reagent of the present invention into contact with a GalNAc donor and a sugar chain represented by the following general formula (1): It is a method for producing a sugar chain represented by the general formula (2).

GlcUA-GalNAc-R ¹ (1) GalNAc-GlcUA-GalNAc-R ¹ (2) (In each formula, -represents a glycoside bond and -R ¹ represents an arbitrary group.)

Specifically, the sugar chain represented by the general formula (1) is preferably a chondroitin polymer or a chondroitin oligomer (eg, hexasaccharide). Also the general formula
Specifically, the sugar chain represented by (2) is Gal at the non-reducing end.
A polymer of chondroitin to which NAc is added or an oligomer of chondroitin (eg, 7-sugar etc.) is preferable.

<6-2> Method 2 for Producing Sugar Chain of the Present Invention Method 2 for producing a sugar chain of the present invention comprises at least a step of bringing the reagent of the present invention into contact with a GalNAc donor and a sugar chain represented by the following general formula (3). And a method for producing a sugar chain represented by the following general formula (4). GlcUA-Gal-R ¹ (3) GalNAc-GlcUA-Gal-R ¹ (4) (In each formula, -represents a glycosidic bond, and -R ¹ represents an arbitrary group.)

Specific examples of the sugar chain represented by the general formula (3) include GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz and the tetrasaccharide (GlcUAβ1-3 of the linkage portion contained in α-thrombomodulin).
Galβ1-3Galβ1-4Xyl) is preferred. Further, as the sugar chain represented by the general formula (4), specifically, GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz having GalNAc added to the non-reducing end and linkage portion 4 having GalNAc added to the non-reducing end can be used. Sugar (GlcUAβ1
-3Galβ1-3Galβ1-4Xyl) is preferred.

GalNAc used in the method for producing a sugar chain of the present invention
As the donor, nucleoside diphosphate-GalNAc is preferable, and UDP-GalNAc is particularly preferable.

The method of "contacting" is based on the protein of the present invention contained in the reagent of the present invention and the donor and acceptor (sugar chain).
There is no particular limitation as long as the above-mentioned molecules come into contact with each other to cause an enzymatic reaction. For example, they may be contacted in a solution in which these three are dissolved. Further, the enzyme of the present invention contained in the reagent of the present invention is bound to an appropriate solid phase (such as beads), or an enzyme is continuously used by using a membrane type reactor using an ultrafiltration membrane, a dialysis membrane or the like. It can also be reacted.
In addition, PCT international publication pamphlet WO00 / 2743
Similar to the method described in No. 7, the receptor can be bound to a solid phase and subjected to an enzymatic reaction. Furthermore, a bioreactor that regenerates (synthesizes) the donor may be used in combination.

The conditions for the enzymatic reaction are not particularly limited as long as they are conditions under which the protein of the present invention (chondroitin synthase) acts.
It is preferable to carry out the reaction at about 6.5), and it is more preferable to carry out the reaction in a buffer solution having a buffer action under the pH. The temperature at this time is not particularly limited as long as the activity of the protein of the present invention is retained,
An example is about -40 ° C (for example, 37 ° C). When there is a substance that increases the activity of the protein of the present invention,
The substance may be added. For example, it is preferable to coexist with Mn ^{2+ and the} like. The reaction time depends on the reagent of the present invention used,
It can be appropriately determined by those skilled in the art depending on the amounts of the donor and the acceptor, and other reaction conditions.

Isolation of chondroitin from the product, etc.
It can be performed by a known method. Further, chondroitin sulfate can also be produced by using the reagent of the present invention (chondroitin synthase) and a sulfate group transferase (sulfotransferase) in combination.

For example, in the above method for producing a sugar chain (method for producing chondroitin), a sulfate group donor (3 '
-Phosphoadenosine 5'-phosphosulfate (PAPS) and a sulfotransferase are allowed to coexist, and chondroitin sulfate can be produced and chondroitin sulfate can be simultaneously transferred to produce chondroitin sulfate. Sulfotransferase may be used as an immobilized enzyme bound to an appropriate solid phase (beads, etc.) in the same manner as described above, and can be continuously treated by using a membrane reactor using an ultrafiltration membrane, a dialysis membrane, etc. May be reacted with. At this time, a bioreactor that regenerates (synthesizes) the sulfate group donor may be used in combination.

In addition, chondroitin can also be produced by directly producing chondroitin in a host transformed with the vector of the present invention (transformant of the present invention).

Further, the vector of the present invention and the cDNA encoding the sulfotransferase are both introduced into a host, and the host (the transformant of the present invention containing the cDNA encoding the sulfotransferase) is treated with chondroitin synthase. A chondroitin sulfate can be directly produced in a host by coexpressing a sulfotransferase.

The sulfate transferase (or cDNA encoding the same) that can be used here is an enzyme that transfers a sulfate group to chondroitin (or cDN encoding the enzyme).
It may be A), and can be appropriately selected from known ones according to the desired type of chondroitin sulfate.
Further, two or more kinds of sulfate group transferases (or cDNAs encoding the same) having different sulfate group transfer positions may be used in combination.

As an example of the sulfate group, for example, chondroitin 6-O-sulfate group (J. Biol. Chem.,
275 (28), 21075-21080 (2000)), but not limited thereto, other enzymes can be used.

<7> Probe of the Present Invention The probe of the present invention is
Nucleotide numbers 655 to 225 in SEQ ID NO: 1
It is a hybridization probe having a nucleotide sequence represented by 0, preferably 778 to 2250, or a sequence complementary to a part thereof.

The probe of the present invention has nucleotide numbers 655 to 2250 in SEQ ID NO: 1, preferably 778 to
It can be obtained by preparing an oligonucleotide having a base sequence represented by 2250 or a sequence complementary to a part thereof, and labeling this with a label suitable for hybridization (eg, radioisotope).

The length of the oligonucleotide is appropriately selected depending on the hybridization conditions using the probe of the present invention.

The probe of the present invention can be used for detecting or quantifying DNA or RNA encoding the protein of the present invention, and is expected to be a useful tool for examining the biological function of chondroitin sulfate. Chondroitin sulfate is
It is widely expressed and plays an important role in many tissues, especially in the brain. The probe may also be useful in exploring the link between genes and disease. Further, the chondroitin synthase 3 will be described in detail in Examples described later.

[0084]

EXAMPLES The present invention will be described in more detail below with reference to examples. <1> Materials and Methods (1) Materials used in this example, etc. UDP- [U- ¹⁴ C] GlcUA (285.2 mCi / mmol) and UDP- [ ³ H] Gal
NAc (10 Ci / mmol) was purchased from NEN Life Science Products. Unlabeled UDP-GlcUA and UDP-GalNAc
Was obtained from Sigma. Chondroitin (chemically desulfated chondroitin sulfate A from whale cartilage), α-N-acetylgalactosaminidase (EC 3.2.1.49) from Acremonium sp., And Arthrobacter aure
Chondroitinase AC-II derived from scens (EC 4.2.2.5)
Was purchased from Seikagaku Corporation. Purified α-thrombomodulin (Reference 17) was provided by the laboratory of Daiichi Pharmaceutical Co., Ltd. For α-thrombomodulin, the tetrasaccharide of the linkage part (GlcUAβ1-3Galβ1-3Galβ1-4Xy
l) is included (Reference 18). The chemically synthesized tetrasaccharide-serine of the linkage part (GlcUAβ1-3Galβ1-3Ga
lβ1-4Xylβ1-O-Ser and GlcUAβ1-3Gal (4-O-sulfate) β1
-3Galβ1-4Xylβ1-O-Ser (Reference 19) was kindly provided by Dr. T. Ogawa of RIKEN. GlcUAβ1-3Galβ1-OC ₂
H ₄ NHCbz was kindly provided by Dr. Junichi Tamura of Tottori University. Chondrohexasaccharide (GlcUAβ1-3GalNAc) ₃ was prepared by the method described in Reference 20. Superdex ^™ Peptide HR10 / 30 columns were purchased from Amersham Pharmacia Biotech.

(2) In silico cloning of human chondroitin synthase 2 Sequence of human chondroitin synthase 1 (Reference 15)
Two new clones were found by tBLASTn analysis of the GenBank ^™ database. One clone (GenBank
^TM accession number: AX092340)
A single open reading frame with significantly higher sequence homology to the carboxyl-terminal side of human chondroitin synthase 1 was shown. In addition, the recently available Human Genome Project database search revealed the same genomic sequence as this cDNA sequence (accession number: NT 029338.1). Comparison of this cDNA with the genomic sequence revealed the genomic organization of the chondroitin synthase 2 gene.

(3) Soluble chondroitin synthase 2
In the chondroitin synthase 2, the first four N-terminals
CD of chondroitin synthase 2 lacking one amino acid
The NA fragment was transferred to G361 human melanoma cells (ATCC C
RL-1424) -derived total RNA as a template, and 5'-primer containing an in-frame BamHI site (5'-CGGGATCCCAGCTG
GCACTGCCCAGG-3 ') (SEQ ID NO: 3) and 58 bp of stop codon
3'-primer (5'-CG containing BamHI site located downstream
GGATCCCATCTCTGACCCATCAGTCC-3 ') (SEQ ID NO: 4) and amplified by reverse transcription-PCR. PCR reaction is 5%
In (v / v) dimethyl sulfoxide, Pfu polymerase (St
ratagene, La Jolla, CA), 94
Thirty cycles of 30 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 180 seconds were performed. This PCR fragment was designated as pGIR201protA.
Subcloned into the BamHI site of Escherichia coli (Reference 21) and GalNA
The c-transferase was fused with the insulin signal sequence and protein A sequence in the vector. An NheI fragment containing the above fusion protein sequence was used as an expression vector.
It was inserted into the XbaI site of pEF-BOS (Reference 22). Amplified c
The nucleotide sequence of DNA was determined using a 377 DNA sequencer (PE Applied Biosystems).

(4) Soluble chondroitin synthase 2
Expression and Enzyme Assay Using FuGENE ^™ 6 (Roche Molecular Biochemicals, Tokyo) according to the manufacturer's instructions, the expression plasmid (6.
7 μg) were transfected into COS-1 cells on 100 mm plates. Two days after transfection, 1 ml of the culture medium was collected and 10 μl of IgG-Sepharose (Amersh
am Pharmacia Biotech) and incubated for 1 hour at 4 ° C. The IgG-Sepharose beads collected by centrifugation were washed with assay buffer, resuspended in the same buffer, and
Used for assay of lNAc transferase and GlcUA transferase activity.

As receptors for measuring GalNAc transferase activity, polymers of chondroitin (167 μg), chondroitin hexasaccharide (GlcUAβ1-3GalNAc) ₃ (10 nmol), α-
Thrombomodulin (1 nmol), GlcUAβ1-3Galβ1-OC ₂ H
₄ NHCbz (1,10, or 100 nmol), GlcUAβ1-3Galβ1-3Ga
lβ1-4Xylβ1-O-Ser (1 nmol) or GlcUAβ1-3Gal (4-O-
Sulfuric acid) β1-3Galβ1-4Xylβ1-O-Ser (1 nmol) was used.

As a receptor for measuring GlcUA transferase activity, a polymer of chondroitin (167 μm) was used.
g) was used.

10 μl of resuspended beads in a total volume of 30 μl as a mixture for the GalNAc transferase assay.
l, receptor substrate 8.57 μM UDP- [ ³ H] GalNAc (3.60 x 10 ⁵ d
pm), 50 mM MES buffer, pH 6.5, 10 mM MnCl ₂ , and A
The one containing 171 μM of TP sodium salt was used (References 23 and 24).

As a mixture for the GlcAT-II assay, 10 μl of resuspended beads, 167 μg, in a total volume of 30 μl
Chondroitin Polymer, 14.3 μM UDP- [ ¹⁴ C] GlcU
^{A (1.46 x 10 5 dpm)} , 50 mM sodium acetate buffer, pH
The one containing 5.6 and 10 mM MnCl ₂ was used (Reference 25).

The reaction mixture was incubated at 37 ° C for 1 hour and the radiolabeled product was subjected to gel filtration using a Sephadex G-25 (Superfine) loaded syringe column or Superdex peptide column. Or Nova-Pak ™ C ₁₈ column (3.9 x 150m
m; Waters), UDP- [ ³ H] GalNAc or UDP
-[ ¹⁴ C] GlcUA separated (Reference 13 and Reference 25 ~
28). The recovered labeled product was quantified by liquid scintillation spectroscopy.

(5) Specific enzyme reaction product The product of the GalNAc transferase reaction using the polymer of chondroitin as an acceptor was isolated by 0.25M NH ₄ HCO ₃ /
Performed by gel filtration using a Superdex peptide column equilibrated with 7% 1-propanol. The radioactive peaks containing each enzymatic reaction product were pooled and evaporated to dryness. This isolated GalNAc transferase reaction product (about 120 μg) was added to 50 mM sodium acetate buffer, pH.
100 mIU overnight at 37 ° C in 30 μl reaction containing 6.0
Digested with chondroitinase AC-II (EC4.2.2.5).
The enzyme digest is the same as the above-mentioned Superdex peptide.
It was analyzed using a column.

Ga using GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz
lNAc transferase reaction product was isolated from HP by combining LC-10A system (Shimadzu) with Nova-Pak (trademark) C ₁₈ column (3.9 x 150 mm; Waters).
Performed by LC. The column was isocratically developed with water at room temperature for 15 minutes at a flow rate of 1.0 ml / min, followed by a linear gradient of 0-100% methanol concentration over 5 minutes, and then the column was isocratically developed with 100% methanol for 40 minutes. did. The radioactive peaks contained in the enzymatic reaction product were pooled and evaporated to dryness. This isolated product (about 74 pmol) was added to 30 μl of a reaction solution containing 50 mM sodium acetate buffer, pH 6.0 at 37 ° C. overnight,
Digested with 100 mIU chondroitinase AC-II, or 50
In a 30 μl reaction solution containing mM sodium acetate buffer, pH 4.5, 40 mIU of α-N-acetylgalactosaminidase was digested at 37 ° C. overnight (Reference 13). Enzyme digests were analyzed using a Nova-Pak ™ C ₁₈ column as described above.

(6) Northern Blot and Expression Array Analysis Commercially available human 12-lane multiple tissue northern blot (manufactured by Clontech) and human multiple tissue expression array (manufactured by Clontech) are used. I was there. The membrane was purified by gel and activated (> 1 × 10 ⁹ cpm / μg) chondroitin synthase 2 cDNA (Genbank accession number: AB071403), nucleotide numbers 698 to 159.
0.95 kb chondroitin synthase 2 specific fragment corresponding to 7 or gel purified and activated (> 1x10 ⁹
cpm / μg) KIAA0990 cDNA (Genbank accession number: AB023207) nucleotide numbers 631 to 1469.
Each of the 0.84 kb fragments specific to chondroitin synthase (Reference 15) was detected as a probe.

<2> Results (1) In silico of human chondroitin synthase 2
In silico cloning We recently identified human chondroitinase synthase (Reference 15), and named it chondroitin synthase 1 this time. National Center for Biotechnology Information
(National Institutes of Health) database,
Screening using the deduced amino acid sequence of human chondroitin synthase 1 revealed a 654 bp 5'-untranslated region,
5 with two N-glycosylated sites (Fig. 1)
A single open reading frame consisting of 1596 bp (nucleotide number 655-2 in SEQ ID NO: 1) encoding a protein consisting of 32 amino acids (SEQ ID NO: 2)
250) and a 1.6 kb 3'-untranslated region with four putative polyadenylation signals (Genbank
Accession number: AX092340) was identified. Northern blot analysis showed that the mRNA length in various human tissues was approximately 4.0 kb, suggesting that the cDNA is almost full length. The deduced amino acid sequence corresponded to the 61350 Da polypeptide.
The predicted translation start site matched the Kozak consensus sequence (Reference 27) and the in-frame stop codon was located upstream of the initiation ATG codon. Kyte-Doolitt
According to le hydropathic analysis (Reference 28), NH ₂ −
One prominent hydrophobic region of 20 amino acid residues in length was shown in the terminal region, indicating that this protein has a type II transmembrane topology of glycosyltransferases that have been cloned to date. Predicted By database search,
Its amino acid sequence has 27% homology to chondroitin synthase 1 (Fig. 1), and human UDP-Gal: Gl
Weak sequence homology was observed with cNAcβ-R β1,4-Galtransferase II (Genbank Accession No .: AB024434), and high COOH-terminal catalytic domain identity was observed. Of note, each protein has a conserved DVD that corresponds to the DXD motif found in many glycosyltransferases (29) (Figure 1). Thus, based on the sequence characteristics of the identified proteins, the identified gene product is β1,4-GalN
It was suggested to have Ac transferase (GalNAcT-I and / or -II) activity. Interestingly, a homologue of the identified human gene was found in Drosophila (Dro
Sophila) but not in the Caenorhabditis elegans genome. The human sequence is Dr
It had 38% identity to osophila.

(2) Genomic organization and chromosomal location As a result of comparing the identified cDNA sequence with the genomic sequence in the Human Genome Project Database, the structure and chromosomal localization of this gene were shown. The gene is 28
Over 0 kb, the coding region of this gene is divided into 7 independent exons as shown in FIG. This intron / exon bond complies with the GT / AG rule (Reference 30), and the surroundings are bound by conserved regions. This gene is present on human chromosome 8.

(3) Expression of Soluble Form of Glycosyltransferase and Properties of Chondroitin Synthase 2 In order to facilitate the functional analysis of GalNAc transferase (chondroitin synthase 2), the soluble form of the protein was glycosylated by the method described above. Replacing the first 41 amino acids of the transferase (amino acids 1-41 in SEQ ID NO: 2) with a cleavable insulin signal and a protein A IgG-binding domain, the soluble glycosyl transferase binds protein A IgG in COS-1 cells. It was produced by expressing as a recombinant enzyme linked to the domain. Expression of an expression plasmid containing a glycosyltransferase / Protein A fusion in COS-1 cells secreted the protein, shown as approximately 95 kDa by Western blotting with IgG. The apparent molecular weight of the fusion protein was reduced to approximately 85 kDa by N-glycosidase treatment. This suggests that both of the two potential N-glycosylation sites are utilized. The fusion enzyme expressed in the culture medium was treated with Ig to remove the endogenous glycosyltransferase.
The beads to which G-sepharose beads were adsorbed and the enzyme was bound were used as an enzyme source. Bound fusion protein,
Glycosyltransferase activity was assayed using various acceptors and UDP-GalNAc or UDP-GlcUA as donor substrates. GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz used as acceptor substrate GalNAcT-I reactions are contained disaccharide in 4 sugar GAG- protein linkage site. As shown in Table 1, remarkable GalNAc transferase activity was observed in the polymer of chondroitin, chondroitin 6
Sugar (GlcUAβ1-3GalNAc) ₃ , a tetrasaccharide (GlcUAβ1-3Galβ1-3Galβ1-3Galβ1 at the linkage site present in the natural core protein)
-4Xylβ1) containing α-thrombomodulin (Reference 18) or GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz as a tetrasaccharide-serine, Glc
UAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser or GlcUAβ1-
It was not found when 3Gal (4-O-sulfate) β1-3Galβ1-4Xylβ1-O-Ser was used as an acceptor substrate.

On the other hand, no GlcUA transferase activity was observed when the chondroitin polymer was used. No detectable glycosyltransferase activity was recovered by affinity purification from control pEF-BOS transfected samples.
These findings clearly indicate that the expressed protein is a GalNAc transferase.

[0100]

[Table 1]

The activity results shown in Table 1 are the average values of two independent experiments. In addition, ND indicates that it was below the detection limit (<0.01 pmol / ml medium / hour).

To identify GalNAc transferase reaction products, each of the typical acceptor substrate polymer of chondroitin and GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz was used as a donor substrate for UDP- [ ³ H] GalNAc. , Labeled by individual transferase reactions using enzyme-coupled beads as the enzyme source. Both labeled products were labeled with β1,4-
It was completely digested with chondroitinase AC-II, which cleaves N-acetylgalactosaminide bond detachably, and subjected to gel filtration (FIG. 3A) or hydrophobic HPLC (FIG. 3B) to obtain free [ ³ H ] Quantitatively produced as a ³ H-labeled peak at the position of GalNAc. This reaction product is also α1,4-catalyzed by EXTL2 (Reference 13).
In contrast to the reaction product of the GalNAc transferase reaction, it was inactive against the reaction of α-N-acetylgalactosaminidase. From these findings, the GalNAc residue is a polymer of chondroitin or GlcUAβ1-3Galβ1-OC ₂ H.
It was shown that ₄ NHCbz was truly transferred to GlcUA at the non-reducing end and bound to β1-4. From these results, the protein identified this time is a novel and unique β that is involved in initiation and elongation of chondroitin / dermatan sulfate biosynthesis.
It was shown to be 1,4-GalNAc transferase I / II. Since the amino acid sequence of this GalNAc transferase has significant homology with chondroitin synthase 1 (FIG. 1), this enzyme was named chondroitin synthase 2.

(4) Expression pattern of chondroitin synthase 2 Northern blot analysis of mRNA revealed a single band of ˜4.0 kb in all human tissues examined this time (FIG. 4). This gene was widely expressed in human tissues examined this time, but to a different extent. It is noted that the expression pattern is similar to that of chondroitin synthase 1.

Array analysis also showed that this gene was expressed in all human tissues examined this time (FIG. 5). The symbols in FIG. 5 are as follows.

A1. Whole brain, B1. Cerebral cortex, C1. Frontal lobe, D1. Parietal lobe, E1. Occipital lobe, F1. Temporal lobe, G1. Paracentral gyrus of cerebral cortex, H1. Bridge.

A2. Left cerebellum, B2. Right cerebellum, C2. Corpus callosum, D2. Tonsils, E2. Caudate nucleus, F2. Hippocampus, G2. Medulla oblongata, H2. Putamen.

B3. Nucleus accumbens, C3. Thalamus.

A4. Heart, B4. Aorta, C4. Left atrium, D4. Right atrium, E4. Left ventricle, F4. Right ventricle, G4. Interventricular nucleus, H4. Apex.

A5. Esophagus, B5. Stomach, C5. Duodenum, D5. Jejunum,
E5. Ileum, F5. Ileocecum, G5. Appendix, H5. Ascending colon.

A6. Transverse colon, B6. Descending colon, C6. Rectum.

A7. Kidney, B7. Skeletal muscle, C7. Spleen, D7. Thymus,
E7. Peripheral blood leukocytes, F7. Lymph glands, G7. Bone marrow, H7. Trachea.

A8. Lung, B8. Placenta, C8. Bladder, D8. Uterus, E8.
Prostate, F8. Testis, G8. Ovary.

A9. Liver, B9. Pancreas, C9. Adrenal gland, D9. Thyroid,
E9. Salivary glands.

A10. Promyelocytic leukemia-derived HL-60 cells, B1
0. Cervical cancer-derived HeLa S3 cells, C10. Chronic myelogenous leukemia-derived K-562 cells, D10. Lymphocytic leukemia-derived MOLT-4 cells, E
10. Burji lymphoma-derived Raji cells, F10. Burkitt lymphoma-derived Daudi cells, G10. Colorectal cancer-derived SW480 cells,
H10. Lung cancer A11. Fetal brain, B11. Fetal heart, C11. Fetal kidney, D11. Fetal liver, E11. Fetal spleen, F11. Fetal thymus, G11. Fetal lung.

A12. Yeast total RNA, B12. Yeast tRNA, C12. E. coli rRNA, D12. E. coli DNA, E12. Polyadenylic acid, F12. Human C0t-1 DNA, G12. Human DNA 100 ng, H12. Human DNA 500
ng.

From FIG. 5, the strongest signals were found in placenta, thyroid, bladder, prostate and adrenal gland. These findings were consistent with the finding that chondroitin / dermatan sulfate proteoglycans are distributed on the surface of many cells and on the extracellular matrix of almost all tissues.

<3> Cloning and activity analysis of a further new chondroitin synthase (chondroitin synthase 3). An attempt was made to clone a new chondroitin synthase (hereinafter referred to as "chondroitin synthase 3").

(1) Cloning of chondroitin synthase 3 Human EST clone KIAA1402 (Kazusa DNA Research Institute (Chiba) HUGE protein database (HYPERLINK http:
//www.kazusa.or.jp/huge/)) (GenBank accession number: AB037823) (SEQ ID NO: 5) was used for cloning according to the following procedure.

5'-agagctcggctagaccaaa as a primer
g-3 '(5'primer; SEQ ID NO: 7) and 5'-ccatcttgccttgcc
A soluble form of KIAA1402 (amino acid numbers 58 to 772 in SEQ ID NO: 6) was amplified by the PCR method using cttcc-3 ′ (3 ′ primer; SEQ ID NO: 8). This was incorporated into pEF-BOS in the same manner as described above to obtain an expression vector. These cloning techniques are the same as described above.

(2) Detection of GlcAT-II activity The expression vector prepared in (1) above was expressed in the same manner as above, and GlcAT-II activity of the expressed protein was measured. The method for measuring GlcAT-II activity is the same as the method described in Reference 15.

GalNAcβ1-4GlcUAβ1-3 as an acceptor substrate
GalNAcβ1-4GlcUAβ1-3GalNAcβ1-4GlcUAβ1-3GalNAc
(Also called chondroitin heptasaccharide) (Preparation method is Kitagaw
a et al., Glycobiology 7, 905-911, 1997) 5 n
Mol was used and incubated overnight at 37 ° C. The reaction product was subjected to gel filtration using Superdex Peptide and measured by liquid scintillation (FIG. 6). As a result, about 2500 dpm was observed (Fig. 6).
Leftmost peak). Since this peak (reaction product) is likely to be cleaved by β-glucuronidase, the protein expressed here is chondroitin and UDP-
It is highly possible that the enzyme has a catalytic activity of transferring GlcUA from GlcUA.

Further, the protein expressed here is Gl
Since the homology with cNAcT-I and GlcNAcT-II is low (Fig. 7), it is considered that they do not have GalNAcT-I and GalNAcT-II activities. According to the method described in Reference 15, GalNA using 1 nmol of α-thrombomodulin as an acceptor substrate
As a result of measuring cT-I activity, this expressed protein (KIAA1402) was considered to have no GalNAcT-I activity.
Therefore, this expressed protein may not have GalNAcT-II activity.

References 1. Kjellen, L., and Lindahl, U. (1991) Annu. Re
v. Biochem. 60, 443-475 2. Salmivirta, M., Lidholt, K., and Lindahl, U.
(1996) FASEB J. 10, 1270-1279 3. Perrimon, N., and Bernfield, M. (2000) Nature
404, 725-728 4. Ohhira, A., Matsui, F., Tokita, Y., Yamauchi,
S., and Aono, S. (2000) Arch. Biochem. Biophys. 3
74, 24-34 5. Sugahara, K., and Yamada, S. (2000) Trends Gl
ycosci. Glycotechnol. 12, 321-349 6. Lindahl, U., and Roden, L. (1972) in Glycopro
teins (Gottschalk, A., ed) pp. 491-517, Elsevier, N
ew York 7. Roden, L. (1980) in The Biochemistry of Glyco
proteins and Proteoglycans (Lennarz, WJ, ed), p
p. 267-371, Plenum Publishing, New York 8. Sugahara, K., and Kitagawa, H. (2000) Curr. O
pin. Struct. Biol. 10,518-527 9. Lind, T., Tufaro, F., McCormick, C., Lindahl,
U., and Lidholt, K. (1998) J. Biol. Chem. 273, 26
265-26268 10. McCormick, C., Duncan, G., Goutsos, KT,
and Tufaro, F. (2000) Proc. Natl. Acad. Sci. US
A. 97, 668-673 11. Senay, C., Lind, T., Muguruma, K., Tone,
Y., Kitagawa, H., Sugahara, K., Lidholt, K., Linda
hl, U., and Kusche-Gullberg, M. (2000) EMBO Report
s 1, 282-286 12. Kim, B.-T., Kitagawa, H., Tamura, J., Sait
o, T., Kusche-Gullberg, M., Lindahl, U., and Sugaha
ra, K. (2001) Proc. Natl. Acad. Sci. USA 98,
7176-7181 13. Kitagawa, H., Shimakawa, H., and Sugahara,
K. (1999) J. Biol. Chem. 274, 13933-13937 14. Fritz, TA, Gabb, MM, Wei, G., and Esk
o, JD (1994) J. Biol. Chem. 269, 28809-28814 15. Kitagawa, H., Uyama, T., and Sugahara, K.
(2001) J. Biol. Chem. 276, 38721-38726 16. Rohrmann, K., Niemann, R., and Buddecke, E.
(1985) Eur. J. Biochem. 148, 463-469 17. Nawa, K., Sakano, K., Fujiwara, H. Sato,
Y., Sugiyama, N., Teruuchi, T., Iwamoto, M., and M
arumoto, Y. (1990) Biochem. Biophys. Res. Commun.
171, 729-737 18. Nadanaka, S., Kitagawa, H., and Sugahara,
K. (1998) J. Biol. Chem. 273, 33728-33734 19. Tamura, J., Neumann, KW, and Ogawa, T. (1
996) Liebigs Ann. 1239-1257 20. Kitagawa, H., Tanaka, Y., Tsuchida, K., Got
o, F., Ogawa, T., Lidholt, K., Lindahl, U., and Su
gahara, K. (1995) J. Biol. Chem. 270, 22190-22195 21. Kitagawa, H., and Paulson, JC (1994) J.
Biol. Chem. 269, 1394-1401 22. Mizushima, S., and Nagata, S. (1990) Nuclei
c Acids Res. 18, 5322 23. Kitagawa, H., Tsuchida, K., Ujikawa, M., an
d Sugahara, K. (1995) J. Biochem. 117, 1083-1087 24. Nadanaka, S., Kitagawa, H., Goto, F., Tamur
a, J., Newmann, KW, Ogawa, T., and Sugahara, K.
(1999) Biochem. J. 340, 353-357 25. Kitagawa, H., Ujikawa, M., Tsutsumi, K., Ta
mura, J., Neumann, KW, Ogawa, T., and Sugahara,
K. (1997) Glycobiology 7, 905-911 26. Kitagawa, H., Tsutsumi, K., Ujikawa, M., Go
to, F., Tamura, J., Neumann, KW, Ogawa, T., and
Sugahara, K. (1997) Glycobiology 7, 531-537 27. Kozak, M. (1984) Nucleic Acids Res. 12, 857
-872 28. Kyte, J., and Doolittle, RF (1982) J. Mo
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u. Rev. Biochem. 50, 349-383

[0124]

INDUSTRIAL APPLICABILITY The protein of the present invention (chondroitin synthase 2) is a novel protein having a specific chondroitin synthase activity and is extremely useful because it can be used in the reagent of the present invention. The vector of the present invention is extremely useful because it can be used for producing the transformant of the present invention. The transformant of the present invention is extremely useful because it can be used in the method for producing the enzyme of the present invention. The enzyme production method of the present invention is extremely useful because it can efficiently produce the protein of the present invention in a large amount. The reagent of the present invention is extremely useful because it can be used as a reagent product or in the method for producing a sugar chain of the present invention. INDUSTRIAL APPLICABILITY The method for producing a sugar chain of the present invention is extremely useful because it can be used for producing a chondroitin or other GalNAc-containing sugar chain. The probe of the present invention is extremely useful because it can be used as a reagent for detecting / quantifying DNA or RNA encoding the protein of the present invention or a useful tool for investigating the relationship between genes and diseases. Further, the probe of the present invention can be used for a microarray of a DNA chip, and is extremely useful. Also, chondroitin synthase 3 is extremely useful as in the above.

[0125]

[Sequence list]                            SEQUENCE LISTING <110> Seikagaku Corporation <120> New chondroitin synthase <130> J200103700 <160> 8 <170> PatentIn Ver. 2.1 <210> 1 <211> 3877 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (655) .. (2253) <400> 1 ctccttaggt ggaaaccctg ggagtagagt actgacagca aagaccggga aagaccatac 60 gtccccgggc aggggtgaca acaggtgtca tctttttgat ctcgtgtgtg gctgccttcc 120 tatttcaagg aaagacgcca aggtaatttt gacccagagg agcaatgatg tagccacctc 180 ctaaccttcc cttcttgaac ccccagttat gccaggattt actagagagt gtcaactcaa 240 ccagcaagcg gctccttcgg cttaacttgt ggttggagga gagaaccttt gtggggctgc 300 gttctcttag cagtgctcag aagtgacttg cctgagggtg gaccagaaga aaggaaaggt 360 cccctcttgc tgttggctgc acatcaggaa ggctgtgatg ggaatgaagg tgaaaacttg 420 gagatttcac ttcagtcatt gcttctgcct gcaagatcat cctttaaaag tagagaagct 480 gctctgtgtg gtggttaact ccaagaggca gaactcgttc tagaaggaaa tggatgcaag 540 cagctccggg ggccccaaac gcatgcttcc tgtggtctag cccagggaag cccttccgtg 600 ggggccccgg ctttgaggga tgccaccggt tctggacgca tggctgattc ctga atg 657                                                             Met                                                               1 atg atg gtt cgc cgg ggg ctg ctt gcg tgg att tcc cgg gtg gtg gtt 705 Met Met Val Arg Arg Gly Leu Leu Ala Trp Ile Ser Arg Val Val Val               5 10 15 ttg ctg gtg ctc ctc tgc tgt gct atc tct gtc ctg tac atg ttg gcc 753 Leu Leu Val Leu Leu Cys Cys Ala Ile Ser Val Leu Tyr Met Leu Ala          20 25 30 tgc acc cca aaa ggt gac gag gag cag ctg gca ctg ccc agg gcc aac 801 Cys Thr Pro Lys Gly Asp Glu Glu Gln Leu Ala Leu Pro Arg Ala Asn      35 40 45 agc ccc acg ggg aag gag ggg tac cag gcc gtc ctt cag gag tgg gag 849 Ser Pro Thr Gly Lys Glu Gly Tyr Gln Ala Val Leu Gln Glu Trp Glu  50 55 60 65 gag cag cac cgc aac tac gtg agc agc ctg aag cgg cag atc gca cag 897 Glu Gln His Arg Asn Tyr Val Ser Ser Leu Lys Arg Gln Ile Ala Gln                  70 75 80 ctc aag gag gag ctg cag gag agg agt gag cag ctc agg aat ggg cag 945 Leu Lys Glu Glu Leu Gln Glu Arg Ser Glu Gln Leu Arg Asn Gly Gln              85 90 95 tac caa gcc agc gat gct gct ggc ctg ggt ctg gac agg agc ccc cca 993 Tyr Gln Ala Ser Asp Ala Ala Gly Leu Gly Leu Asp Arg Ser Pro Pro         100 105 110 gag aaa acc cag gcc gac ctc ctg gcc ttc ctg cac tcg cag gtg gac 1041 Glu Lys Thr Gln Ala Asp Leu Leu Ala Phe Leu His Ser Gln Val Asp     115 120 125 aag gca gag gtg aat gct ggc gtc aag ctg gcc aca gag tat gca gca 1089 Lys Ala Glu Val Asn Ala Gly Val Lys Leu Ala Thr Glu Tyr Ala Ala 130 135 140 145 gtg cct ttc gat agc ttt act cta cag aag gtg tac cag ctg gag act 1137 Val Pro Phe Asp Ser Phe Thr Leu Gln Lys Val Tyr Gln Leu Glu Thr                 150 155 160 ggc ctt acc cgc cac ccc gag gag aag cct gtg agg aag gac aag cgg 1185 Gly Leu Thr Arg His Pro Glu Glu Lys Pro Val Arg Lys Asp Lys Arg             165 170 175 gat gag ttg gtg gaa gcc att gaa tca gcc ttg gag acc ctg aac aat 1233 Asp Glu Leu Val Glu Ala Ile Glu Ser Ala Leu Glu Thr Leu Asn Asn         180 185 190 cct gca gag aac agc ccc aat cac cgt cct tac acg gcc tct gat ttc 1281 Pro Ala Glu Asn Ser Pro Asn His Arg Pro Tyr Thr Ala Ser Asp Phe     195 200 205 ata gaa ggg atc tac cga aca gaa agg gac aaa ggg aca ttg tat gag 1329 Ile Glu Gly Ile Tyr Arg Thr Glu Arg Asp Lys Gly Thr Leu Tyr Glu 210 215 220 225 ctc acc ttc aaa ggg gac cac aaa cac gaa ttc aaa cgg ctc atc tta 1377 Leu Thr Phe Lys Gly Asp His Lys His Glu Phe Lys Arg Leu Ile Leu                 230 235 240 ttt cga cca ttc agc ccc atc atg aaa gtg aaa aat gaa aag ctc aac 1425 Phe Arg Pro Phe Ser Pro Ile Met Lys Val Lys Asn Glu Lys Leu Asn             245 250 255 atg gcc aac acg ctt atc aat gtt atc gtg cct cta gca aaa agg gtg 1473 Met Ala Asn Thr Leu Ile Asn Val Ile Val Pro Leu Ala Lys Arg Val         260 265 270 gac aag ttc cgg cag ttc atg cag aat ttc agg gag atg tgc att gag 1521 Asp Lys Phe Arg Gln Phe Met Gln Asn Phe Arg Glu Met Cys Ile Glu     275 280 285 cag gat ggg aga gtc cat ctc act gtt gtt tac ttt ggg aaa gaa gaa 1569 Gln Asp Gly Arg Val His Leu Thr Val Val Tyr Phe Gly Lys Glu Glu 290 295 300 305 ata aat gaa gtc aaa gga ata ctt gaa aac act tcc aaa gct gcc aac 1617 Ile Asn Glu Val Lys Gly Ile Leu Glu Asn Thr Ser Lys Ala Ala Asn                 310 315 320 ttc agg aac ttt acc ttc atc cag ctg aat gga gaa ttt tct cgg gga 1665 Phe Arg Asn Phe Thr Phe Ile Gln Leu Asn Gly Glu Phe Ser Arg Gly             325 330 335 aag gga ctt gat gtt gga gcc cgc ttc tgg aag gga agc aac gtc ctt 1713 Lys Gly Leu Asp Val Gly Ala Arg Phe Trp Lys Gly Ser Asn Val Leu         340 345 350 ctc ttt ttc tgt gat gtg gac atc tac ttc aca tct gaa ttc ctc aat 1761 Leu Phe Phe Cys Asp Val Asp Ile Tyr Phe Thr Ser Glu Phe Leu Asn     355 360 365 acg tgt agg ctg aat aca cag cca ggg aag aag gta ttt tat cca gtt 1809 Thr Cys Arg Leu Asn Thr Gln Pro Gly Lys Lys Val Phe Tyr Pro Val 370 375 380 385 ctt ttc agt cag tac aat cct ggc ata ata tac ggc cac cat gat gca 1857 Leu Phe Ser Gln Tyr Asn Pro Gly Ile Ile Tyr Gly His His Asp Ala                 390 395 400 gtc cct ccc ttg gaa cag cag ctg gtc ata aag aag gaa act gga ttt 1905 Val Pro Pro Leu Glu Gln Gln Leu Val Ile Lys Lys Glu Thr Gly Phe             405 410 415 tgg aga gac ttt gga ttt ggg atg acg tgt cag tat cgg tca gac ttc 1953 Trp Arg Asp Phe Gly Phe Gly Met Thr Cys Gln Tyr Arg Ser Asp Phe         420 425 430 atc aat ata ggt ggg ttt gat ctg gac atc aaa ggc tgg ggc gga gag 2001 Ile Asn Ile Gly Gly Phe Asp Leu Asp Ile Lys Gly Trp Gly Gly Glu     435 440 445 gat gtg cac ctt tat cgc aag tat ctc cac agc aac ctc ata gtg gta 2049 Asp Val His Leu Tyr Arg Lys Tyr Leu His Ser Asn Leu Ile Val Val 450 455 460 465 cgg acg cct gtg cga gga ctc ttc cac ctc tgg cat gag aag cgc tgc 2097 Arg Thr Pro Val Arg Gly Leu Phe His Leu Trp His Glu Lys Arg Cys                 470 475 480 atg gac gag ctg acc ccc gag cag tac aag atg tgc atg cag tcc aag 2145 Met Asp Glu Leu Thr Pro Glu Gln Tyr Lys Met Cys Met Gln Ser Lys             485 490 495 gcc atg aac gag gca tcc cac ggc cag ctg ggc atg ctg gtg ttc agg 2193 Ala Met Asn Glu Ala Ser His Gly Gln Leu Gly Met Leu Val Phe Arg         500 505 510 cac gag ata gag gct cac ctt cgc aaa cag aaa cag aag aca agt agc 2241 His Glu Ile Glu Ala His Leu Arg Lys Gln Lys Gln Lys Thr Ser Ser     515 520 525 aaa aaa aca tga actcccagag aaggattgtg ggagacactt tttctttcct 2293 Lys Lys Thr 530 tttgcaatta ctgaaagtgg ctgcaacaga gaaaagactt ccataaagga cgacaaaaga 2353 attggactga tgggtcagag atgagaaagc ctccgatttc tctctgttgg gctttttaca 2413 acagaaatca aaatctccgc tttgcctgca aaagtaaccc agttgcaccc tgtgaagtgt 2473 ctgacaaagg cagaatgctt gtgagattat aagcctaatg gtgtggaggt tttgatggtg 2533 tttacaatac actgagacct gttgtttttt gtgctcattg aaatattcat gatttaagag 2593 cagttttgta aaaaattcat tagcatgaaa ggcaagcata tttctcctca tatgaatgag 2653 cctatcagca gggctctagt ttctaggaat gctaaaatat cagaaggcag gagaggagat 2713 aggcttatta tgatactagt gagtacatta agtaaaataa aatggaccag aaaagaaaag 2773 aaaccataaa tatcgtgtca tattttcccc aagattaacc aaaaataatc tgcttatctt 2833 tttggttgtc cttttaactg tctccgtttt tttcttttat ttaaaaatgc actttttttc 2893 ccttgtgagt tatagtctgc ttatttaatt accactttgc aagccttaca agagagcaca 2953 agttggccta catttttata ttttttaaga agatactttg agatgcatta tgagaacttt 3013 cagttcaaag catcaaattg atgccatatc caaggacatg ccaaatgctg attctgtcag 3073 gcactgaatg tcaggcattg agacataggg aaggaatggt ttgtactaat acagacgtac 3133 agatactttc tctgaagagt attttcgaag aggagcaact gaacactgga ggaaaagaaa 3193 atgacacttt ctgctttaca gaaaaggaaa ctcattcaga ctggtgatat cgtgatgtac 3253 ctaaaagtca gaaaccacat tttctcctca gaagtaggga ccgctttctt acctgtttaa 3313 ataaaccaaa gtataccgtg tgaaccaaac aatctctttt caaaacaggg tgctcctcct 3373 ggcttctggc ttccataaga agaaatggag aaaaatatat atatatatat atatattgtg 3433 aaagatcaat ccatctgcca gaatctagtg ggatggaagt ttttgctaca tgttatccac 3493 cccaggccag gtggaagtaa ctgaattatt ttttaaatta agcagttcta ctcaatcacc 3553 aagatgcttc tgaaaattgc attttattac catttcaaac tattttttaa aaataaatac 3613 agttaacata gagtggtttc ttcattcatg tgaaaattat tagccagcac cagatgcatg 3673 agctaattat ctctttgagt ccttgcttct gtttgctcac agtaaactca ttgtttaaaa 3733 gcttcaagaa cattcaagct gttggtgtgt taaaaaatgc attgtattga tttgtactgg 3793 tagtttatga aatttaatta aaacacaggc catgaatgga aggtggtatt gcacagctaa 3853 taaaatatga tttgtggata tgaa 3877 <210> 2 <211> 532 <212> PRT <213> Homo sapiens <400> 2 Met Met Met Val Arg Arg Gly Leu Leu Ala Trp Ile Ser Arg Val Val   1 5 10 15 Val Leu Leu Val Leu Leu Cys Cys Ala Ile Ser Val Leu Tyr Met Leu              20 25 30 Ala Cys Thr Pro Lys Gly Asp Glu Glu Gln Leu Ala Leu Pro Arg Ala          35 40 45 Asn Ser Pro Thr Gly Lys Glu Gly Tyr Gln Ala Val Leu Gln Glu Trp      50 55 60 Glu Glu Gln His Arg Asn Tyr Val Ser Ser Leu Lys Arg Gln Ile Ala  65 70 75 80 Gln Leu Lys Glu Glu Leu Gln Glu Arg Ser Glu Gln Leu Arg Asn Gly                  85 90 95 Gln Tyr Gln Ala Ser Asp Ala Ala Gly Leu Gly Leu Asp Arg Ser Pro             100 105 110 Pro Glu Lys Thr Gln Ala Asp Leu Leu Ala Phe Leu His Ser Gln Val         115 120 125 Asp Lys Ala Glu Val Asn Ala Gly Val Lys Leu Ala Thr Glu Tyr Ala     130 135 140 Ala Val Pro Phe Asp Ser Phe Thr Leu Gln Lys Val Tyr Gln Leu Glu 145 150 155 160 Thr Gly Leu Thr Arg His Pro Glu Glu Lys Pro Val Arg Lys Asp Lys                 165 170 175 Arg Asp Glu Leu Val Glu Ala Ile Glu Ser Ala Leu Glu Thr Leu Asn             180 185 190 Asn Pro Ala Glu Asn Ser Pro Asn His Arg Pro Tyr Thr Ala Ser Asp         195 200 205 Phe Ile Glu Gly Ile Tyr Arg Thr Glu Arg Asp Lys Gly Thr Leu Tyr     210 215 220 Glu Leu Thr Phe Lys Gly Asp His Lys His Glu Phe Lys Arg Leu Ile 225 230 235 240 Leu Phe Arg Pro Phe Ser Pro Ile Met Lys Val Lys Asn Glu Lys Leu                 245 250 255 Asn Met Ala Asn Thr Leu Ile Asn Val Ile Val Pro Leu Ala Lys Arg             260 265 270 Val Asp Lys Phe Arg Gln Phe Met Gln Asn Phe Arg Glu Met Cys Ile         275 280 285 Glu Gln Asp Gly Arg Val His Leu Thr Val Val Tyr Phe Gly Lys Glu     290 295 300 Glu Ile Asn Glu Val Lys Gly Ile Leu Glu Asn Thr Ser Lys Ala Ala 305 310 315 320 Asn Phe Arg Asn Phe Thr Phe Ile Gln Leu Asn Gly Glu Phe Ser Arg                 325 330 335 Gly Lys Gly Leu Asp Val Gly Ala Arg Phe Trp Lys Gly Ser Asn Val             340 345 350 Leu Leu Phe Phe Cys Asp Val Asp Ile Tyr Phe Thr Ser Glu Phe Leu         355 360 365 Asn Thr Cys Arg Leu Asn Thr Gln Pro Gly Lys Lys Val Phe Tyr Pro     370 375 380 Val Leu Phe Ser Gln Tyr Asn Pro Gly Ile Ile Tyr Gly His His Asp 385 390 395 400 Ala Val Pro Pro Leu Glu Gln Gln Leu Val Ile Lys Lys Glu Thr Gly                 405 410 415 Phe Trp Arg Asp Phe Gly Phe Gly Met Thr Cys Gln Tyr Arg Ser Asp             420 425 430 Phe Ile Asn Ile Gly Gly Phe Asp Leu Asp Ile Lys Gly Trp Gly Gly         435 440 445 Glu Asp Val His Leu Tyr Arg Lys Tyr Leu His Ser Asn Leu Ile Val     450 455 460 Val Arg Thr Pro Val Arg Gly Leu Phe His Leu Trp His Glu Lys Arg 465 470 475 480 Cys Met Asp Glu Leu Thr Pro Glu Gln Tyr Lys Met Cys Met Gln Ser                 485 490 495 Lys Ala Met Asn Glu Ala Ser His Gly Gln Leu Gly Met Leu Val Phe             500 505 510 Arg His Glu Ile Glu Ala His Leu Arg Lys Gln Lys Gln Lys Thr Ser         515 520 525 Ser Lys Lys Thr     530 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer for PCR       (5 'primer) <400> 3 cgggatccca gctggcactg cccagg 26 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer for       PCR (3 'primer) <400> 4 cgggatccca tctctgaccc atcagtcc 28 <210> 5 <211> 3970 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1514) .. (3832) <400> 5 aggggctgtg aggtggcagc ggctgcagcg gcggagccgg cgcctcagcg ggcactgggg 60 tctgttcccc cttccccgtc cctgctccct gccaggcgcg tgcgggacgc cgctcttggt 120 ccccacgcct ccgccccgcc ccctcccggg acgccgggag accccggccg tcctttatcc 180 ggtgtccgcc ggcccccggc cctgaaaccc gggcctcctc cccgagggcc ttgggcgtcc 240 ctcctggtcc tgcgctcgcg gcctcgatgc tgtctctggc gcggcctccg ctcccgccga 300 ctggcctgag aacgaggtct gtgccccagt ctcccagccg cgacctccga ccccgcctcg 360 cagaacgacc cgagctggtc tcccgagccc ccttctcagc agcccggtga cgtggccagt 420 ttatttctgt tttgagacga acggcgaaga ctcgcgtcgg gtcttcgttc tagggctaga 480 cttggtctct gatcgccgag aggtagcgca ggggctgtgg gcccccggca ggggtcctgt 540 cggaagctgg ccgcgcttct gtttgcgttc ccaggaccct ggcattgtct ctagttgctg 600 cttgtgctct ctctttgctt ttggtttgct tcatttggcc cctggggccc tggtaaaacc 660 aggcaccgaa tcgctcgcac acagagttcc agtccccgcc tgctgtctcc tcagcagctg 720 gggttccgag gagaatgccc tgcaagatgg ctccatcggc catggcgctc ctgagaggct 780 gtcagtgctg agtcaccgat ctacctcatt cgggtgggca gaacttatgt gtgccatgcg 840 agtggctcca gaccgctcct gagtgggagg aggggttcct gtagccgttg cgtcttctca 900 aacacgggga gcagagtaga aagaggctct ggccccttcc cttgtgccca ccgggcctgc 960 cgcagtggct cagcagcccc ttcagtagcc cgcctgagga ccgatgccag aggcaggcat 1020 tcttccggaa aggcccactg aggcaggctc cggctcctct ggttggggct gttgttttga 1080 tggatcgtgt gcttttccct tacctcttat cacttgctgt catctgttga cttaggccca 1140 gtctgcagat gtgtgtagtg ttcctttttg ggttagcttt ggcagtattg agttttactt 1200 cctcctcttt ttagtggaag acagaccata atcccagtgt gagtgaaatt gattgtttca 1260 tttattaccg ttttggctgg gggttagttc cgacaccttc acagttgaag agcaggcaga 1320 aggagttgtg aagacaggac aatcttcttg gggatgctgg tcctggaagc cagcgggcct 1380 cgctctgtct ttggcctcat tgaccccagg ttctctggtt aaaactgaaa gcctactact 1440 ggcctggtgc ccatcaatcc attgatcctt gaggctgtgc ccctggggca cccacctggc 1500 agggcctacc acc atg cga ctg agc tcc ctg ttg gct ctg ctg cgg cca 1549                Met Arg Leu Ser Ser Leu Leu Ala Leu Leu Arg Pro                  1 5 10 gcg ctt ccc ctc atc tta ggg ctg tct ctg ggg tgc agc ctg agc ctc 1597 Ala Leu Pro Leu Ile Leu Gly Leu Ser Leu Gly Cys Ser Leu Ser Leu          15 20 25 ctg cgg gtt tcc tgg atc cag ggg gag gga gaa gat ccc tgt gtc gag 1645 Leu Arg Val Ser Trp Ile Gln Gly Glu Gly Glu Asp Pro Cys Val Glu      30 35 40 gct gta ggg gag cga gga ggg cca cag aat cca gat tcc aga gct cgg 1693 Ala Val Gly Glu Arg Gly Gly Pro Gln Asn Pro Asp Ser Arg Ala Arg  45 50 55 60 cta gac caa agt gat gaa gac ttc aaa ccc cgg att gtc ccc tac tac 1741 Leu Asp Gln Ser Asp Glu Asp Phe Lys Pro Arg Ile Val Pro Tyr Tyr                  65 70 75 agg gac ccc aac aag ccc tac aag aag gtg ctc agg act cgg tac atc 1789 Arg Asp Pro Asn Lys Pro Tyr Lys Lys Val Leu Arg Thr Arg Tyr Ile              80 85 90 cag aca gag ctg ggc tcc cgt gag cgg ttg ctg gtg gct gtc ctg acc 1837 Gln Thr Glu Leu Gly Ser Arg Glu Arg Leu Leu Val Ala Val Leu Thr          95 100 105 tcc cga gct aca ctg tcc act ttg gcc gtg gct gtg aac cgt acg gtg 1885 Ser Arg Ala Thr Leu Ser Thr Leu Ala Val Ala Val Asn Arg Thr Val     110 115 120 gcc cat cac ttc cct cgg tta ctc tac ttc act ggg cag cgg ggg gcc 1933 Ala His His Phe Pro Arg Leu Leu Tyr Phe Thr Gly Gln Arg Gly Ala 125 130 135 140 cgg gct cca gca ggg atg cag gtg gtg tct cat ggg gat gag cgg ccc 1981 Arg Ala Pro Ala Gly Met Gln Val Val Ser His Gly Asp Glu Arg Pro                 145 150 155 gcc tgg ctc atg tca gag acc ctg cgc cac ctt cac aca cac ttt ggg 2029 Ala Trp Leu Met Ser Glu Thr Leu Arg His Leu His Thr His Phe Gly             160 165 170 gcc gac tac gac tgg ttc ttc atc atg cag gat gac aca tat gtg cag 2077 Ala Asp Tyr Asp Trp Phe Phe Ile Met Gln Asp Asp Thr Tyr Val Gln         175 180 185 gcc ccc cgc ctg gca gcc ctt gct ggc cac ctc agc atc aac caa gac 2125 Ala Pro Arg Leu Ala Ala Leu Ala Gly His Leu Ser Ile Asn Gln Asp     190 195 200 ctg tac tta ggc cgg gca gag gag ttc att ggc gca ggc gag cag gcc 2173 Leu Tyr Leu Gly Arg Ala Glu Glu Phe Ile Gly Ala Gly Glu Gln Ala 205 210 215 220 cgg tac tgt cat ggg ggc ttt ggc tac ctg ttg tca cgg agt ctc ctg 2221 Arg Tyr Cys His Gly Gly Phe Gly Tyr Leu Leu Ser Arg Ser Leu Leu                 225 230 235 ctt cgt ctg cgg cca cat ctg gat ggc tgc cga gga gac att ctc agt 2269 Leu Arg Leu Arg Pro His Leu Asp Gly Cys Arg Gly Asp Ile Leu Ser             240 245 250 gcc cgt cct gac gag tgg ctt gga cgc tgc ctc att gac tct ctg ggc 2317 Ala Arg Pro Asp Glu Trp Leu Gly Arg Cys Leu Ile Asp Ser Leu Gly         255 260 265 gtc ggc tgt gtc tca cag cac cag ggg cag cag tat cgc tca ttt gaa 2365 Val Gly Cys Val Ser Gln His Gln Gly Gln Gln Tyr Arg Ser Phe Glu     270 275 280 ctg gcc aaa aat agg gac cct gag aag gaa ggg agc tcg gct ttc ctg 2413 Leu Ala Lys Asn Arg Asp Pro Glu Lys Glu Gly Ser Ser Ala Phe Leu 285 290 295 300 agt gcc ttc gcc gtg cac cct gtc tcc gaa ggt acc ctc atg tac cgg 2461 Ser Ala Phe Ala Val His Pro Val Ser Glu Gly Thr Leu Met Tyr Arg                 305 310 315 ctc cac aaa cgc ttc agc gct ctg gag ttg gag cgg gct tac agt gaa 2509 Leu His Lys Arg Phe Ser Ala Leu Glu Leu Glu Arg Ala Tyr Ser Glu             320 325 330 ata gaa caa ctg cag gct cag atc cgg aac ctg acc gtg ctg acc ccc 2557 Ile Glu Gln Leu Gln Ala Gln Ile Arg Asn Leu Thr Val Leu Thr Pro         335 340 345 gaa ggg gag gca ggg ctg agc tgg ccc gtt ggg ctc cct gct cct ttc 2605 Glu Gly Glu Ala Gly Leu Ser Trp Pro Val Gly Leu Pro Ala Pro Phe     350 355 360 aca cca cac tct cgc ttt gag gtg ctg ggc tgg gac tac ttc aca gag 2653 Thr Pro His Ser Arg Phe Glu Val Leu Gly Trp Asp Tyr Phe Thr Glu 365 370 375 380 cag cac acc ttc tcc tgt gca gat ggg gct ccc aag tgc cca cta cag 2701 Gln His Thr Phe Ser Cys Ala Asp Gly Ala Pro Lys Cys Pro Leu Gln                 385 390 395 ggg gct agc agg gcg gac gtg ggt gat gcg ttg gag act gcc ctg gag 2749 Gly Ala Ser Arg Ala Asp Val Gly Asp Ala Leu Glu Thr Ala Leu Glu             400 405 410 cag ctc aat cgg cgc tat cag ccc cgc ctg cgc ttc cag aag cag cga 2797 Gln Leu Asn Arg Arg Tyr Gln Pro Arg Leu Arg Phe Gln Lys Gln Arg         415 420 425 ctg ctc aac ggc tat cgg cgc ttc gac cca gca cgg ggc atg gag tac 2845 Leu Leu Asn Gly Tyr Arg Arg Phe Asp Pro Ala Arg Gly Met Glu Tyr     430 435 440 acc ctg gac ctg ctg ttg gaa tgt gtg aca cag cgt ggg cac cgg cgg 2893 Thr Leu Asp Leu Leu Leu Glu Cys Val Thr Gln Arg Gly His Arg Arg 445 450 455 460 gcc ctg gct cgc agg gtc agc ctg ctg cgg cca ctg agc cgg gtg gaa 2941 Ala Leu Ala Arg Arg Val Ser Leu Leu Arg Pro Leu Ser Arg Val Glu                 465 470 475 atc cta cct atg ccc tat gtc act gag gcc acc cga gtg cag ctg gtg 2989 Ile Leu Pro Met Pro Tyr Val Thr Glu Ala Thr Arg Val Gln Leu Val             480 485 490 ctg cca ctc ctg gtg gct gaa gct gct gca gcc ccg gct ttc ctc gag 3037 Leu Pro Leu Leu Val Ala Glu Ala Ala Ala Ala Pro Ala Phe Leu Glu         495 500 505 gcc ttt gca gcc aat gtc ctg gag cca cga gaa cat gca ttg ctc acc 3085 Ala Phe Ala Ala Asn Val Leu Glu Pro Arg Glu His Ala Leu Leu Thr     510 515 520 ctg ttg ctg gtc tac ggg cca cga gaa ggt ggc cgt gga gct cca gac 3133 Leu Leu Leu Val Tyr Gly Pro Arg Glu Gly Gly Arg Gly Ala Pro Asp 525 530 535 540 cca ttt ctt ggg gtg aag gct gca gca gcg gag tta gag cga cgg tac 3181 Pro Phe Leu Gly Val Lys Ala Ala Ala Ala Glu Leu Glu Arg Arg Tyr                 545 550 555 cct ggg acg agg ctg gcc tgg ctc gct gtg cga gca gag gcc cct tcc 3229 Pro Gly Thr Arg Leu Ala Trp Leu Ala Val Arg Ala Glu Ala Pro Ser             560 565 570 cag gtg cga ctc atg gac gtg gtc tcg aag aag cac cct gtg gac act 3277 Gln Val Arg Leu Met Asp Val Val Ser Lys Lys His Pro Val Asp Thr         575 580 585 ctc ttc ttc ctt acc acc gtg tgg aca agg cct ggg ccc gaa gtc ctc 3325 Leu Phe Phe Leu Thr Thr Val Trp Thr Arg Pro Gly Pro Glu Val Leu     590 595 600 aac cgc tgt cgc atg aat gcc atc tct ggc tgg cag gcc ttc ttt cca 3373 Asn Arg Cys Arg Met Asn Ala Ile Ser Gly Trp Gln Ala Phe Phe Pro 605 610 615 620 gtc cat ttc cag gag ttc aat cct gcc ctg tca cca cag aga tca ccc 3421 Val His Phe Gln Glu Phe Asn Pro Ala Leu Ser Pro Gln Arg Ser Pro                 625 630 635 cca ggg ccc ccg ggg gct ggc cct gac ccc ccc tcc cct cct ggt gct 3469 Pro Gly Pro Pro Gly Ala Gly Pro Asp Pro Pro Ser Pro Pro Gly Ala             640 645 650 gac ccc tcc cgg ggg gct cct ata ggg ggg aga ttt gac cgg cag gct 3517 Asp Pro Ser Arg Gly Ala Pro Ile Gly Gly Arg Phe Asp Arg Gln Ala         655 660 665 tct gcg gag ggc tgc ttc tac aac gct gac tac ctg gcg gcc cga gcc 3565 Ser Ala Glu Gly Cys Phe Tyr Asn Ala Asp Tyr Leu Ala Ala Arg Ala     670 675 680 cgg ctg gca ggt gaa ctg gca ggc cag gaa gag gag gaa gcc ctg gag 3613 Arg Leu Ala Gly Glu Leu Ala Gly Gln Glu Glu Glu Glu Ala Leu Glu 685 690 695 700 ggg ctg gag gtg atg gat gtt ttc ctc cgg ttc tca ggg ctc cac ctc 3661 Gly Leu Glu Val Met Asp Val Phe Leu Arg Phe Ser Gly Leu His Leu                 705 710 715 ttt cgg gcc gta gag cca ggg ctg gtg cag aag ttc tcc ctg cga gac 3709 Phe Arg Ala Val Glu Pro Gly Leu Val Gln Lys Phe Ser Leu Arg Asp             720 725 730 tgc agc cca cgg ctc agt gaa gaa ctc tac cac cgc tgc cgc ctc agc 3757 Cys Ser Pro Arg Leu Ser Glu Glu Leu Tyr His Arg Cys Arg Leu Ser         735 740 745 aac ctg gag ggg cta ggg ggc cgt gcc cag ctg gct atg gct ctc ttt 3805 Asn Leu Glu Gly Leu Gly Gly Arg Ala Gln Leu Ala Met Ala Leu Phe     750 755 760 gag cag gag cag gcc aat agc act tag cccgcctggg ggccctaacc 3852 Glu Gln Glu Gln Ala Asn Ser Thr 765 770 tcattacctt tcctttgtct gcctcagccc caggaagggc aaggcaagat ggtggacaga 3912 tagagaattg ttgctgtatt ttttaaatat gaaaatgtta ttaaacatgt cttctgcc 3970 <210> 6 <211> 772 <212> PRT <213> Homo sapiens <400> 6 Met Arg Leu Ser Ser Leu Leu Ala Leu Leu Arg Pro Ala Leu Pro Leu   1 5 10 15 Ile Leu Gly Leu Ser Leu Gly Cys Ser Leu Ser Leu Leu Arg Val Ser              20 25 30 Trp Ile Gln Gly Glu Gly Glu Asp Pro Cys Val Glu Ala Val Gly Glu          35 40 45 Arg Gly Gly Pro Gln Asn Pro Asp Ser Arg Ala Arg Leu Asp Gln Ser      50 55 60 Asp Glu Asp Phe Lys Pro Arg Ile Val Pro Tyr Tyr Arg Asp Pro Asn  65 70 75 80 Lys Pro Tyr Lys Lys Val Leu Arg Thr Arg Tyr Ile Gln Thr Glu Leu                  85 90 95 Gly Ser Arg Glu Arg Leu Leu Val Ala Val Leu Thr Ser Arg Ala Thr             100 105 110 Leu Ser Thr Leu Ala Val Ala Val Asn Arg Thr Val Ala His His Phe         115 120 125 Pro Arg Leu Leu Tyr Phe Thr Gly Gln Arg Gly Ala Arg Ala Pro Ala     130 135 140 Gly Met Gln Val Val Ser His Gly Asp Glu Arg Pro Ala Trp Leu Met 145 150 155 160 Ser Glu Thr Leu Arg His Leu His Thr His Phe Gly Ala Asp Tyr Asp                 165 170 175 Trp Phe Phe Ile Met Gln Asp Asp Thr Tyr Val Gln Ala Pro Arg Leu             180 185 190 Ala Ala Leu Ala Gly His Leu Ser Ile Asn Gln Asp Leu Tyr Leu Gly         195 200 205 Arg Ala Glu Glu Phe Ile Gly Ala Gly Glu Gln Ala Arg Tyr Cys His     210 215 220 Gly Gly Phe Gly Tyr Leu Leu Seru Arg Ser Leu Leu Leu Arg Leu Arg 225 230 235 240 Pro His Leu Asp Gly Cys Arg Gly Asp Ile Leu Ser Ala Arg Pro Asp                 245 250 255 Glu Trp Leu Gly Arg Cys Leu Ile Asp Ser Leu Gly Val Gly Cys Val             260 265 270 Ser Gln His Gln Gly Gln Gln Tyr Arg Ser Phe Glu Leu Ala Lys Asn         275 280 285 Arg Asp Pro Glu Lys Glu Gly Ser Ser Ala Phe Leu Ser Ala Phe Ala     290 295 300 Val His Pro Val Ser Glu Gly Thr Leu Met Tyr Arg Leu His Lys Arg 305 310 315 320 Phe Ser Ala Leu Glu Leu Glu Arg Ala Tyr Ser Glu Ile Glu Gln Leu                 325 330 335 Gln Ala Gln Ile Arg Asn Leu Thr Val Leu Thr Pro Glu Gly Glu Ala             340 345 350 Gly Leu Ser Trp Pro Val Gly Leu Pro Ala Pro Phe Thr Pro His Ser         355 360 365 Arg Phe Glu Val Leu Gly Trp Asp Tyr Phe Thr Glu Gln His Thr Phe     370 375 380 Ser Cys Ala Asp Gly Ala Pro Lys Cys Pro Leu Gln Gly Ala Ser Arg 385 390 395 400 Ala Asp Val Gly Asp Ala Leu Glu Thr Ala Leu Glu Gln Leu Asn Arg                 405 410 415 Arg Tyr Gln Pro Arg Leu Arg Phe Gln Lys Gln Arg Leu Leu Asn Gly             420 425 430 Tyr Arg Arg Phe Asp Pro Ala Arg Gly Met Glu Tyr Thr Leu Asp Leu         435 440 445 Leu Leu Glu Cys Val Thr Gln Arg Gly His Arg Arg Ala Leu Ala Arg     450 455 460 Arg Val Ser Leu Leu Arg Pro Leu Ser Arg Val Glu Ile Leu Pro Met 465 470 475 480 Pro Tyr Val Thr Glu Ala Thr Arg Val Gln Leu Val Leu Pro Leu Leu                 485 490 495 Val Ala Glu Ala Ala Ala Ala Pro Ala Phe Leu Glu Ala Phe Ala Ala             500 505 510 Asn Val Leu Glu Pro Arg Glu His Ala Leu Leu Thr Leu Leu Leu Val         515 520 525 Tyr Gly Pro Arg Glu Gly Gly Arg Gly Ala Pro Asp Pro Phe Leu Gly     530 535 540 Val Lys Ala Ala Ala Ala Glu Leu Glu Arg Arg Tyr Pro Gly Thr Arg 545 550 555 560 Leu Ala Trp Leu Ala Val Arg Ala Glu Ala Pro Ser Gln Val Arg Leu                 565 570 575 Met Asp Val Val Ser Lys Lys His Pro Val Asp Thr Leu Phe Phe Leu             580 585 590 Thr Thr Val Trp Thr Arg Pro Gly Pro Glu Val Leu Asn Arg Cys Arg         595 600 605 Met Asn Ala Ile Ser Gly Trp Gln Ala Phe Phe Pro Val His Phe Gln     610 615 620 Glu Phe Asn Pro Ala Leu Ser Pro Gln Arg Ser Pro Pro Gly Pro Pro 625 630 635 640 Gly Ala Gly Pro Asp Pro Pro Ser Pro Pro Gly Ala Asp Pro Ser Arg                 645 650 655 Gly Ala Pro Ile Gly Gly Arg Phe Asp Arg Gln Ala Ser Ala Glu Gly             660 665 670 Cys Phe Tyr Asn Ala Asp Tyr Leu Ala Ala Arg Ala Arg Leu Ala Gly         675 680 685 Glu Leu Ala Gly Gln Glu Glu Glu Glu Ala Leu Glu Gly Leu Glu Val     690 695 700 Met Asp Val Phe Leu Arg Phe Ser Gly Leu His Leu Phe Arg Ala Val 705 710 715 720 Glu Pro Gly Leu Val Gln Lys Phe Ser Leu Arg Asp Cys Ser Pro Arg                 725 730 735 Leu Ser Glu Glu Leu Tyr His Arg Cys Arg Leu Ser Asn Leu Glu Gly             740 745 750 Leu Gly Gly Arg Ala Gln Leu Ala Met Ala Leu Phe Glu Gln Glu Gln         755 760 765 Ala Asn Ser Thr     770 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer for PCR       (5 'primer) <400> 7 agagctcggc tagaccaaag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer for PCR       (3 'primer) <400> 8 ccatcttgcc ttgcccttcc 20

[Brief description of drawings]

FIG. 1 Human-derived chondroitin synthase 2 (chondro)
The comparison of the deduced amino acid sequences of itin synthase 2) and human-derived chondroitin synthase 1 is shown. These deduced amino acid sequences are GENETYX-MA
It was analyzed using the C (version 10) computer program. Black boxes indicate that the amino acids are identical between them. The gap introduced to maximize the degree of agreement is shown by the dashed line. The predicted transmembrane domain is boxed. The conserved DXD motif is underlined. Two possible sites of N-glycosylation sites of human-derived chondroitin synthase 2 are marked with asterisks.

FIG. 2 shows the genomic organization of the chondroitin synthase 2 gene. The exon region is boxed. Black boxes show coding sequences, white boxes show 5'- and 3'-
The untranslated sequence is shown. The translation initiation codon (ATG) and stop codon (TGA) are also shown. Black horizontal lines indicate introns.

FIG. 3 shows the results of identifying chondroitin synthase 2 reaction products. The vertical axis represents radioactivity ( ³ H-Radioactivity), and the horizontal axis represents fraction number. (A) The ³ H-labeled GalNAc transferase II reaction product obtained by using a chondroitin polymer as an acceptor substrate was treated with chondroitinase by the method described in Examples.
Digested with AC-II. Chondroitinase AC-II digested product (black circles) or undigested product (black triangles) was applied to the Superdex peptide column (1.0 x 30 cm), and for each elution fraction (0.4 ml each), Radioactivity was analyzed by the method described in the example. The arrow indicates the elution position of free GalNAc. (B) The ³ H-labeled GalNAc transferase I reaction product obtained using GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz as an acceptor substrate was treated with chondroitinase AC-II or α- by the method described in Example. Digested with N-acetylgalactosaminidase. Chondroitinase AC-II digested products (black circles), α-N-acetylgalactosaminidase digested products (black squares) or undigested products (black triangles) were treated with Nova- by the method described in Examples. HPLC analysis using a Pak (trade name) C ₁₈ column was performed, and each elution fraction (2 ml each) was analyzed for radioactivity. The arrow indicates the elution position of free GalNAc.

FIG. 4 Chondroitin synthase 2 in human tissues
And the results of Northern blot analysis of chondroitin synthase 1. Hybridization was performed on RNA derived from various human tissues by the method described in Examples using a probe of chondroitin synthase 2 (upper row in the figure) or chondroitin synthase 1 (lower row in the figure). Lane 1
Is brain, lane 2 is heart, lane 3 is skeletal muscle, lane 4 is colon, lane 5 is thymus, lane 6 is spleen, lane 7 is kidney, lane 8 is liver, lane 9 is small intestine, lane 10 is placenta, lane 11 is a lung, and lane 12 is a peripheral blood leukocyte.

FIG. 5 shows the results of RNA dot blot analysis of chondroitin synthase 2 and chondroitin synthase 1. Human Multiple Tissue Expression Array (Clontech), chondroitin synthase 2 (upper figure) or chondroitin synthase 1
Hybridization was carried out by the method described in Examples using the probe (lower part of the figure).

FIG. 6 shows the results of identification of reaction products of chondroitin synthase 3. Vertical axis shows radioactivity ( ¹⁴ C-Radioactivity)
The abscissa indicates the fraction number.

FIG. 7: Human chondroitin synthase 1 (chondro)
The comparison of the deduced amino acid sequences of itin synthase 1), human-derived chondroitin synthase 2 (chondroitin synthase 2), and human-derived chondroitin synthase 3 (chondroitin synthase 3) is shown. The analysis was performed as in FIG.
Black boxes indicate that the amino acids are identical between them. The gap introduced to maximize the degree of agreement is shown by the dashed line. Also, the saved DXD motif is surrounded by a square frame.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 5/10 C12P 19/14 Z 9/10 C12N 15/00 ZNAA C12P 19/14 5/00 A F term (Reference) 4B024 AA03 BA10 CA04 CA12 4B050 CC03 DD11 LL01 LL05 4B064 AF04 CA19 CC24 CD09 CD12 CE10 DA01 4B065 AA93Y AB01 BA01 CA29 CA44 4H045 AA11 BA10 CA40 DA89 EA20 FA74

Claims (41)

[Reference number] J200103700 [Claims] 1. A protein of the following (A) or (B): (A) A protein containing the amino acid sequence represented by amino acid numbers 42 to 532 in SEQ ID NO: 2. (B) A protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or transposed in the amino acid sequence of (A) above and having the following properties (a) and (b). (A) It has a catalytic activity for transferring GalNAc from UDP-GalNAc to chondroitin. (UDP represents uridine 5'-diphosphate and GalNAc represents an N-acetylgalactosamine residue.) (B) It has substantially no catalytic activity for transferring GlcUA from UDP-GlcUA to chondroitin. (UDP is Uridine 5'-
For diphosphate, GlcUA indicates a glucuronic acid residue. ) 2. In addition to the above (a) and (b), it further has the following properties (c) and (d):
The protein according to claim 1. (C) From α-thrombomodulin to UDP-GalNAc to GalN
It has catalytic activity to transfer Ac. (UDP is Uridine 5'-
Diphosphate and GalNAc represent N-acetylgalactosamine residues. (D) It has catalytic activity for transferring GalNAc from UDP-GalNAc to GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz. (UDP represents uridine 5'-diphosphate, GlcUA represents a glucuronic acid residue, Gal represents a galactose residue, GalNAc represents an N-acetylgalactosamine residue, and Cbz represents a benzyloxycarbonyl group.) 3. The protein of (A) is SEQ ID NO: 2.
The protein according to claim 1 or 2, which is a protein consisting of the amino acid sequence represented by amino acid numbers 42 to 532 in. 4. The protein of (A) is SEQ ID NO: 2.
The protein according to claim 1 or 2, which is a fusion protein of the protein consisting of the amino acid sequence represented by amino acid Nos. 42 to 532 in 1 above and another peptide or polypeptide. 5. The protein of (A) is SEQ ID NO: 2.
The protein according to claim 1 or 2, which is a protein consisting of the amino acid sequence represented by amino acid Nos. 1 to 532. 6. The protein according to any one of claims 1 to 5, wherein the protein is a soluble protein. 7. A DNA according to any one of the following (a) to (c)
Holding vector. (A) D encoding a protein containing the amino acid sequence represented by amino acid numbers 42 to 532 in SEQ ID NO: 2
NA. (B) The amino acid sequence represented by amino acid numbers 42 to 532 in SEQ ID NO: 2 contains an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or transposed, and has the following properties (a) and (b): DNA encoding a protein having: (C) hybridizes with the DNA described in (a) above, a DNA complementary to the DNA, or a DNA having a part of the base sequence of these DNAs under stringent conditions, and (a) and A DNA encoding a protein having the property (b). (A) It has a catalytic activity for transferring GalNAc from UDP-GalNAc to chondroitin. (UDP represents uridine 5'-diphosphate and GalNAc represents an N-acetylgalactosamine residue.) (B) It has substantially no catalytic activity for transferring GlcUA from UDP-GlcUA to chondroitin. (UDP is Uridine 5'-
For diphosphate, GlcUA indicates a glucuronic acid residue. ) 8. The vector according to claim 7, which further comprises a DNA encoding a protein having the following properties (c) and (d) in addition to (a) and (b): . (C) From α-thrombomodulin to UDP-GalNAc to GalN
It has catalytic activity to transfer Ac. (UDP is Uridine 5'-
Diphosphate and GalNAc represent N-acetylgalactosamine residues. (D) It has catalytic activity for transferring GalNAc from UDP-GalNAc to GlcUAβ1-3Galβ1-OC ₂ H ₄ NHCbz. (UDP represents uridine 5'-diphosphate, GlcUA represents a glucuronic acid residue, Gal represents a galactose residue, GalNAc represents an N-acetylgalactosamine residue, and Cbz represents a benzyloxycarbonyl group.) 9. The DNA of (a) above has amino acid number 4
The vector according to claim 7 or 8, which is a DNA encoding a protein consisting of the amino acid sequences represented by 2 to 532. 10. A DNA encoding a protein having an amino acid sequence represented by amino acid numbers 42 to 532.
Are nucleotide numbers 778 to 22 in SEQ ID NO: 1.
The vector according to claim 9, which is the DNA represented by 50. 11. The DNA according to claim 7 or 8, wherein the DNA (a) is a DNA encoding a fusion protein of a protein having the amino acid sequence represented by amino acid numbers 42 to 532 and another peptide or polypeptide. The described vector. 12. A DNA encoding a fusion protein of a protein consisting of the amino acid sequence represented by amino acid numbers 42 to 532 and another peptide or polypeptide,
Nucleotide numbers 778 to 2250 in SEQ ID NO: 1
The vector according to claim 11, which is a ligation product of the DNA shown in (4) and a DNA encoding another peptide or polypeptide. 13. The vector according to claim 7, wherein the DNA of (a) is a DNA encoding a protein having an amino acid sequence represented by amino acid numbers 1 to 532. 14. A DNA encoding a protein consisting of the amino acid sequences represented by amino acid numbers 1 to 532,
Nucleotide number 655 to 2250 in SEQ ID NO: 1
The vector according to claim 13, which is a DNA represented by: 15. The vector according to any one of claims 7 to 14, wherein the protein is a soluble protein. 16. The expression vector according to claim 7, which is an expression vector.
The vector according to any one of 1. 17. A transformant obtained by transforming a host with the vector according to any one of claims 7 to 16. 18. A method for producing chondroitin synthase, which comprises growing the transformant according to claim 17 and collecting chondroitin synthase from the grown product. 19. A N-acetylgalactosamine-containing sugar chain synthesizing reagent, which comprises the protein according to any one of claims 1 to 6. 20. Production of a sugar chain represented by the following general formula (2), which comprises at least a step of contacting the reagent according to claim 19 with a GalNAc donor and a sugar chain represented by the following general formula (1). Method. GlcUA-GalNAc-R ¹ (1) GalNAc-GlcUA-GalNAc-R ¹ (2) (In each formula, GlcUA and GalNAc have the same meanings as described above.-Represents a glycosidic bond and -R ¹ represents an arbitrary Indicates a group.) 21. Production of a sugar chain represented by the following general formula (4), which comprises at least a step of contacting the reagent according to claim 19 with a GalNAc donor and a sugar chain represented by the following general formula (3). Method. GlcUA-Gal-R ¹ (3) GalNAc-GlcUA-Gal-R ¹ (4) (In each formula, GlcUA, GalNAc and Gal are as defined above.- is a glycosidic bond and -R ¹ is Indicates any group.) 22. A hybridization probe having a sequence complementary to the nucleotide sequence represented by nucleotide numbers 655 to 2250 in SEQ ID NO: 1 or a part thereof. 23. The following protein (A) or (B): (A) A protein comprising the amino acid sequence represented by amino acid numbers 58 to 772 in SEQ ID NO: 6. (B) A protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or transposed in the amino acid sequence of (A) above and having the following properties (a) and (b). (A) UDP-GlcU is added to the chondroitin heptasaccharide shown below.
It has catalytic activity to transfer GlcUA from A. GalNAc- (GlcUAβ1-3GalNAc) ₃ (UDP indicates uridine 5′-diphosphate, GlcUA indicates glucuronic acid residue, GalNAc indicates N-acetylgalactosamine residue.) (B) From chondroitin to UDP-GalNAc It has substantially no catalytic activity to transfer GalNAc. (UDP is Uridine 5 '
-Diphosphate, GalNAc represents an N-acetylgalactosamine residue. ) 24. The protein according to claim 23, wherein the protein (A) is a protein having an amino acid sequence represented by amino acid numbers 58 to 772 in SEQ ID NO: 6. 25. The protein according to claim 23, wherein the protein (A) is a fusion protein of a protein consisting of the amino acid sequence represented by amino acid numbers 58 to 772 in SEQ ID NO: 6 and another peptide or polypeptide. protein. 26. The protein according to claim 23, wherein the protein (A) is a protein having an amino acid sequence represented by amino acid numbers 1 to 772 in SEQ ID NO: 6. 27. The protein according to any one of claims 23 to 26, wherein the protein is a soluble protein. 28. The DN according to any one of the following (a) to (c)
A vector carrying A. (A) D encoding a protein containing the amino acid sequence represented by amino acid numbers 58 to 772 in SEQ ID NO: 6
NA. (B) the amino acid sequence represented by amino acid numbers 58 to 772 in SEQ ID NO: 2 contains an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or transposed, and has the following properties (a) and (b) DNA encoding a protein having: (C) hybridizes with the DNA described in (a) above, a DNA complementary to the DNA, or a DNA having a part of the base sequence of these DNAs under stringent conditions, and (a) and A DNA encoding a protein having the property (b). (A) UDP-GlcU is added to the chondroitin heptasaccharide shown below.
It has catalytic activity to transfer GlcUA from A. GalNAc- (GlcUAβ1-3GalNAc) ₃ (UDP indicates uridine 5′-diphosphate, GlcUA indicates glucuronic acid residue, GalNAc indicates N-acetylgalactosamine residue.) (B) From chondroitin to UDP-GalNAc It has substantially no catalytic activity to transfer GalNAc. (UDP is Uridine 5 '
-Diphosphate, GalNAc represents an N-acetylgalactosamine residue. ) 29. The vector according to claim 28, wherein the DNA of (a) is a DNA encoding a protein having an amino acid sequence represented by amino acid numbers 58 to 772. 30. A DNA encoding a protein having an amino acid sequence represented by amino acid numbers 58 to 772.
Is nucleotide number 1685-3 in SEQ ID NO: 5
The vector according to claim 29, which is the DNA represented by 829. 31. The DNA according to claim 28, wherein the DNA of (a) is a DNA encoding a fusion protein of a protein consisting of the amino acid sequence represented by amino acid numbers 58 to 772 and another peptide or polypeptide. vector. 32. A DNA encoding a fusion protein of a protein consisting of the amino acid sequence represented by amino acid numbers 58 to 772 and another peptide or polypeptide,
Nucleotide number 1685-382 in SEQ ID NO: 1
The vector according to claim 31, which is a ligation product of the DNA shown in 9 and a DNA encoding another peptide or polypeptide. 33. The vector according to claim 28, wherein the DNA of (a) is a DNA encoding a protein having an amino acid sequence represented by amino acid numbers 1 to 772. 34. A DNA encoding a protein consisting of the amino acid sequence represented by amino acid numbers 1 to 772,
Nucleotide numbers 1514 to 382 in SEQ ID NO: 5
34. The vector according to claim 33, which is the DNA represented by 9. 35. The vector according to any one of claims 28 to 34, wherein the protein is a soluble protein. 36. An expression vector according to claims 28 to 3.
The vector according to any one of 5 above. 37. A transformant obtained by transforming a host with the vector according to any one of claims 28 to 36. 38. A method for producing chondroitin synthase, which comprises growing the transformant according to claim 37 and collecting chondroitin synthase from the grown product. 39. A glucuronic acid-containing sugar chain synthesizing reagent containing the protein according to any one of claims 23 to 27. 40. Production of a sugar chain represented by the following general formula (6), which comprises at least a step of contacting the reagent according to claim 39 with a GlcUA donor and a sugar chain represented by the following general formula (5). Method. GalNAc-R ¹ (5) GlcUA-GalNAc-R ¹ (6) (In each formula, GlcUA and GalNAc have the same meanings as described above.-Represents a glycosidic bond and -R ¹ represents an arbitrary group. ) 41. A hybridization probe having a base sequence represented by nucleotide numbers 1514 to 3829 in SEQ ID NO: 5 or a sequence complementary to a part thereof.