JPH10257896A - Polypeptide of glycosaminoglycan sulfate group transferase and dna coding for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new DNA coding for the polypeptide of a specific sulfate group transferase having a specific amino acid sequence and capable of transferring the sulfate group from a sulfuric acid donor to glycosaminoglycan, and useful for producing the enzyme used for clarifying the structure-function relation of sulfated polysaccharide, etc. SOLUTION: This new DNA has a base sequence coding for at least one portion of a glycosaminoglycan sulfate group transferase polypeptide which has an amino acid sequence of the formula, can convert a sulfate group from a sulfate group donor to the 2-hydroxyl group of L-iduronic acid residue contained in a glycosaminoglycan of sulfate group receptor and may contain one or more amino acid residues substantially not damaging the activity of the enzyme, and is useful for clarifying the structure-function relation of the sulfated polysaccharide, etc. The DNA is obtained by extracting mRNA from the ovarian cells of a Chinese hamster, making up a cDNA library from the mRNA, and subsequently screening the library with a partial DNA fragment of glycosaminoglycan sulfate group transferase as a probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a polypeptide of glycosaminoglycan sulfotransferase (glycosaminoglycan sulfotransferase) and a novel DNA having a nucleotide sequence encoding the same. More specifically, 2 of L-iduronic acid contained in heparan sulfate
The present invention relates to a polypeptide of heparan sulfate 2-O-sulfotransferase derived from Chinese hamsters and humans, which selectively sulfates the hydroxyl group at position 2, and a DNA having a base sequence encoding the polypeptide. Further, the present invention provides the DNA
The present invention relates to a method for producing a glycosaminoglycan sulfotransferase polypeptide using the same.

[0002]

2. Description of the Related Art Heparan sulfate is known as hexuronic acid (Hex).
A) Residue (D-glucuronic acid (GlcA) residue or L
-A repeating structure of disaccharide of iduronic acid (IdoA) residue and N-acetylglucosamine residue (GlcNAc) (4
GlcAβ1 / IdoAα1 → 4GlcNAcα1) as the basic skeleton, and a glycosaminoglycan having a sulfate group at each of a part of the 2-position of the hexuronic acid residue and a part of the 2- and 6-position of the N-acetylglucosamine residue. It is a kind.

[0003] By cloning the gene for glycosaminoglycan sulfotransferase, it becomes possible to obtain information on the substrate specificity of glycosaminoglycan as a sulfate group acceptor, and to obtain glycosaminoglycan. It will provide a useful approach to study the relationship between glycan structure and function. It is known that the biosynthesis of glycosaminoglycans, and especially the biosynthesis of heparin / heparan sulfate, involves many sulfation processes (Yo Kiwata, Senichiro Hakomori, Katsutaka Nagai ed., Glycotechnology, p. 57). , 1994, published by Kodansha Scientific), it is thought that various glycosaminoglycan sulfate transferases are involved in this sulfation. Glycosaminoglycan sulfate transferases that transfer sulfate groups to heparin / heparan sulfate include heparan sulfate 2-O-sulfate transferase (hereinafter sometimes abbreviated as "HS2ST") and heparan sulfate 6-O. -A sulfotransferase (hereinafter sometimes abbreviated as "HS6ST") has been isolated. However, cloning of cDNA is difficult.

[0004] The present inventors have already found that the sulfate group donor 3 '
-Heparan sulfate 2-O-sulfate group transfer, which selectively transfers a sulfate group from phosphoadenosine 5'-phosphosulfate to a hydroxyl group at position 2 of an L-iduronic acid residue contained in heparan sulfate which is a sulfate group acceptor. The enzyme is purified from cultured cells (CHO cells) derived from Chinese hamster ovary (Ko
bayashi, M., Habuchi, H., Habuchi, O., Saito, M.,
and Kimata, K. (1996) J. Biol. Chem. 271, 7645-765.
3). However, the cloning of the enzyme has not yet been performed. In addition, human-derived heparan sulfate 2-O
-Sulfotransferase and the DNA encoding it have not yet been obtained.

[0005]

It is important in structural analysis of heparan sulfate to obtain a large amount of an enzyme which selectively transfers a sulfate group to a hydroxyl group at the 2-position of L-iduronic acid residue contained in heparan sulfate. Cloning of the cDNA for the enzyme is very important, as it will provide a means. That is, the present invention provides a means for obtaining the enzyme in a large amount by a simple method by cloning the polypeptide encoding the enzyme and the cDNA encoding the sequence thereof, whereby the structure-function relationship of the sulfated polysaccharide is provided. The purpose is to contribute to the elucidation of.

[0006]

Means for Solving the Problems The present inventors have proposed heparan sulfate or N, O-desulfated re-N-sulfated heparin (also referred to herein as "CDSNS-heparin").
DNA encoding a glycosaminoglycan sulfate transferase that selectively transfers a sulfate group to the hydroxyl group at position 2 of the L-iduronic acid residue contained in the above, ie, a polypeptide of heparan sulfate 2-O-sulfate transferase The present inventors completed the present invention by confirming that the cDNA having the nucleotide sequence encoding the polypeptide of the enzyme was successfully cloned and that the cDNA expressed heparan sulfate 2-O-sulfotransferase. .

That is, the present invention has an amino acid sequence represented by SEQ ID NO: 14 and converts a sulfate group from a sulfate group donor to a 2-position of L-iduronic acid residue contained in glycosaminoglycan which is a sulfate group acceptor. Polypeptide of glycosaminoglycan sulfate transferase which may have substitution, deletion, insertion or rearrangement of one or more amino acid residues which does not substantially impair the enzymatic activity of selectively transferring to a hydroxyl group of A DNA having a base sequence encoding at least a portion of

The DNA of the present invention includes a DNA having a base sequence encoding at least a part of the amino acid sequence shown in SEQ ID NO: 14, and more specifically, at least a part of the amino acid sequence shown in SEQ ID NO: 2. And a DNA having a base sequence encoding at least a part of the amino acid sequence shown in SEQ ID NO: 4.

The present invention also provides a DNA having a base sequence encoding a sulfotransferase polypeptide having the following properties. Action: A sulfate group is selectively transferred from a sulfate group donor to a hydroxyl group at the 2-position of an L-iduronic acid residue contained in glycosaminoglycan which is a sulfate group acceptor. Substrate specificity: transfers sulfate groups to heparan sulfate and CDSNS-heparin, but does not transfer sulfate groups to chondroitin, chondroitin sulfate, dermatan sulfate and keratan sulfate. Optimum reaction pH: around pH 5.0 to 6.5 Optimum reaction ionic strength: around 50 to 200 mM (in the case of sodium chloride) Inhibition and activation Enzyme activity is increased by coexisting protamine. Adenosine-3 ', 5'-diphosphate (3', 5'-A
Enzyme activity is inhibited by the coexistence of DP).
Coexistence of 10 mM or less of dithiothreitol (DTT) hardly affects the enzyme activity. Molecular weight: molecular weight estimated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions after N-glycosidase treatment: about 38,000.

Further, the present invention provides a polypeptide comprising all or a part of the glycosaminoglycan sulfotransferase polypeptide encoded by the above DNA base sequence.

Furthermore, the present invention provides an amino acid sequence represented by SEQ ID NO: 4, wherein a sulfate group is converted from a sulfate group donor to an L-amino acid contained in glycosaminoglycan which is a sulfate group acceptor.
Substitution or deletion of one or more amino acid residues that do not substantially impair the enzymatic activity of transferring to the hydroxyl group at position 2 of the iduronic acid residue,
A glycosaminoglycan sulfotransferase comprising a polypeptide which may have an insertion or transposition is provided.

The glycosaminoglycan sulfate transferase (hereinafter also referred to as "the enzyme") containing the polypeptide encoded by the nucleotide sequence of the DNA of the present invention is conveniently referred to as a heparan sulfate 2-O-sulfate group. Although referred to as transferase or heparan sulfate 2-O-sulfotransferase, this does not mean that the substrate for the enzyme is limited to heparan sulfate. For example, the present enzyme is a chemically modified heparin obtained by re-N-sulfating N, O-desulfated heparin (N, O-desulfated re-N-sulfated heparin. -Heparin ”).
Sulfate group is also selectively transferred to the hydroxyl group at the position. Unmodified heparin has a sulfate group at position 2 of most L-iduronic acid residues, but some have a slight hydroxyl group, and the enzyme encoded by the nucleotide sequence of the DNA of the present invention is The sulfate group is also selectively transferred to the hydroxyl group at the 2-position of such L-iduronic acid residue of heparin. In the present specification, modified heparin such as CDSNS-heparin may be simply referred to as heparin.

Further, the present invention provides a method for culturing cells transformed with the above DNA in a suitable medium to produce and accumulate in the culture a polypeptide of the present enzyme encoded by the base sequence of the DNA. And a method for producing a polypeptide, comprising collecting the polypeptide from a culture thereof.

[0014]

Embodiments of the present invention will be described below. <1> DNA having a nucleotide sequence encoding a polypeptide of glycosaminoglycan sulfate transferase of the present invention (DNA of the present invention) The DNA of the present invention has an amino acid sequence shown in SEQ ID NO: 14, and is derived from a sulfate group donor. The sulfate group is replaced with two L-iduronic acid residues contained in glycosaminoglycan which is a sulfate group acceptor.
Polysaccharide of glycosaminoglycan sulfate transferase which may have substitution, deletion, insertion or rearrangement of one or more amino acid residues which does not substantially impair the enzymatic activity of selectively transferring to the hydroxyl group at position 1. Provided is a DNA having a nucleotide sequence encoding at least a part of a peptide.

The glycosaminoglycan sulfotransferase containing the polypeptide encoded by the nucleotide sequence of the DNA of the present invention can be obtained by converting L-iduronic acid contained in glycosaminoglycan, which is a sulfate group acceptor, from a sulfate group donor. Heparan sulfate 2-O-sulfotransferase which selectively transfers a sulfate group to a hydroxyl group at the 2-position of a residue.

The DNA of the present invention is a DNA isolated for the first time by the present invention, and encodes at least a part of a polypeptide of heparan sulfate 2-O-sulfotransferase (hereinafter also referred to as "HS2ST"). If so, the base sequence is not particularly limited. Further, the HS2ST polypeptide encoded by the nucleotide sequence of the DNA of the present invention can be obtained by converting a sulfate group from a sulfate group donor to a hydroxyl group at the 2-position of an L-iduronic acid residue contained in glycosaminoglycan which is a sulfate group acceptor. May have one or more amino acid residue substitutions, deletions, insertions, or transpositions that do not substantially impair the activity of selective transfer to the DNA of the present invention. Included. Methods for measuring the activity are known.
(For example, J. Biol. Chem. 271, 7645-7653 (1996)), those skilled in the art can use one or more amino acid residues that do not substantially impair the target enzyme activity as an indicator. The present enzyme having a substitution, deletion, insertion or transposition of a group in an amino acid sequence can be easily selected.

In the amino acid sequence shown in SEQ ID NO: 14, amino acid No. 39 is preferably a neutral amino acid, more preferably serine or alanine, and amino acid No. 67 is preferably a neutral amino acid, more preferably threonine or alanine. Yes, amino acid number 68 is preferably a hydrophobic amino acid, more preferably leucine or valine, amino acid number 74 is preferably methionine or isoleucine, and amino acid number 100 is preferably a basic amino acid, more preferably Lysine or arginine, amino acid number 130 is preferably a hydroxyl group-containing amino acid, more preferably serine or threonine, amino acid number 132 is preferably lysine or asparagine, and amino acid number 142 is preferably a hydrophobic amino acid. , More preferably valine or isoleucine, amino acid number 277 is preferably acidic amino acid, preferably glutamic acid or aspartic acid. Here, the basic amino acid preferably means histidine, lysine and arginine, the acidic amino acid preferably means glutamic acid and aspartic acid, and the neutral amino acid is an amino acid that is neither acidic nor basic. That is, an amino acid having no charge in a normal biological environment, preferably glycine, alanine, serine, threonine, and the hydrophobic amino acid is glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, It means cysteine and amino acids having the same hydrophobicity, preferably valine, leucine and isoleucine.

In the above DNA of the present invention, SEQ ID NO: 14
Is preferably the amino acid sequence shown in SEQ ID NO: 2 or 4. In addition, the DNA of the present invention preferably encodes a nucleotide sequence encoding at least a part of the amino acid sequence shown in SEQ ID NO: 14, 2 or 4 without substitution, deletion, insertion or transposition of amino acid residues.

Specifically, the DNA of the present invention includes amino acids 1 to 35 in the amino acid sequence shown in SEQ ID NO: 14.
DNA having a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 6; more specifically, the amino acid sequence represented by amino acid numbers 1-356 in SEQ ID NO: 2 and amino acids 1-356 in SEQ ID NO: 4 A DNA having an encoding base sequence is exemplified and preferred.
More specifically, the base sequence of the DNA of the present invention is a DNA having at least a part or all of the base sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3, and is particularly preferable. Specific examples of such DNA include base numbers 24 to 1091 in the base sequence shown in SEQ ID NO: 1.
And base number 355 in the base sequence shown in SEQ ID NO: 3.
DNA having a base sequence of 1422.

In the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3, four in-frame ATs are located at the 5 'end of the open reading frame of HS2ST cDNA.
Contains G codons. Compared with the consensus sequence of the translation initiation site in eukaryotic cells, the nucleotide sequence around the first ATG codon does not preserve the purine at position -3, but preserves G (guanine) at position +4. Have been. This satisfies Kozak's finding (Kozak, M. (1986) Cell, 44,283-292) that for efficient translation, G at +4 is essential when no purine is at -3. doing. Also, in the nucleotide sequence around the second, third and fourth ATG codons, the position of -3 is purine (each A
(Adenine), A, G), and +4 is not G, but A, C (cytosine), C, respectively, and partially conforms to the consensus sequence, and any ATG codon functions as an initiation codon there is a possibility. However, since the fourth ATG codon corresponds to the hydrophobic site of the transmembrane region in the amino acid sequence, it is unlikely to function as a translation initiation site.

By the way, it is known that β-1,4-galactosyltransferase contains two ATG codons in frame (Nakazawa, K. et al. (1988)).
J. Biochem, 104, 165-168, Shaper, N. et al. (1988)
J. Biol. Chem., 263, 10420-10428). Also, Shaper
Have shown that β-1,4-galactosyltransferase is synthesized in both long and short forms as a result of initiation of translation from two sites. Furthermore, Lopez et al. Show evidence suggesting that long forms preferentially target the plasma membrane, while shorter forms are predominantly in the Golgi (Lopez, L. et a
l. (1991) J. Biol. Chem., 266, 15984-15591). Similarly, for HS2ST, a plurality of ATG codons may function as initiation codons, but it is not clear.
However, no matter which ATG codon is the initiation codon, the same is true in that it encodes the above-mentioned sulfotransferase polypeptide, and the second and third nucleotides in the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 3 A DNA having a base sequence starting from the fourth ATG codon is also included in the present invention.

A single open reading frame starting at the first ATG codon of SEQ ID NO: 1 consists of 356 amino acid residues, a molecular weight of 41,830, and two potential N-linked glycosylation sites. A protein having a site is predicted. From the hydropathy plot created from this amino acid sequence (FIG. 2), N-
One prominent hydrophobic portion of 14 residues in length spanning the 14th to 27th amino acid residues from the end was observed, and is expected to have a transmembrane (transmembrane) domain.
In addition, a single open reading frame starting with the first ATG codon of SEQ ID NO: 3 is composed of 356 amino acid residues, has a molecular weight of 41,868, and has two potential N-linked glycosylation sites. Is predicted. From the hydropathy plot created from this amino acid sequence, 14-27 from the N-terminal
One prominent hydrophobic portion of 14 residues in length spanning the second amino acid residue is observed and is expected to have a transmembrane (transmembrane) domain.

The DNA of the present invention is characterized in that the HS2ST polypeptide encoded by the nucleotide sequence contained in the DNA is obtained by converting a sulfate group from a sulfate group donor to an L-iduronic acid residue contained in heparan sulfate which is a sulfate group acceptor. Unless the activity of selectively transferring to the 2-position hydroxyl group is substantially impaired.
It may have nucleotide substitutions, deletions, insertions or transpositions that cause substitution, deletion, insertion or transposition of one or more amino acid residues. Nucleotide substitution, deletion, insertion or transposition has a restriction enzyme cleaved end at both ends, synthesizes a sequence containing both sides of the mutation point, and replaces the corresponding portion of the base sequence of the unmutated DNA, thereby obtaining DNA Can be introduced. Site-directed mutagenesis (Kramer, W. and Frits, HJ, Meth. In Enzymo
l., 154, 350 (1987); Kunkel, TA et al., Meth. in En
zymol., 154, 367 (1987)).
Substitutions, deletions, insertions or transpositions can be introduced into
Methods for measuring the activity of the present enzyme are known (for example, J. Bio
l. Chem. 271, 7645-7653 (1996)), and those skilled in the art can use one or more amino acid residues as indicators of the substitution or deletion of one or more amino acid residues that do not substantially impair the activity. D encoding the present enzyme having an amino acid sequence of deletion, insertion or transposition
Substitution, deletion, insertion or transposition of the base sequence in NA can be easily selected.

It should be readily understood by those skilled in the art that DNAs having different base sequences due to the degeneracy of the genetic code are also included in the DNA of the present invention. Furthermore, the HS2ST gene derived from the chromosome is expected to contain an intron in the coding region.
As long as it encodes at least a part of the ST polypeptide, it is included in the DNA fragment of the present invention. That is, in the present specification, the term “encode” also includes having a base sequence that can be processed during transcription to finally produce a target polypeptide.

In the present specification, “encoding at least a portion of a polypeptide” is preferably HS.
A part having 2ST activity or having any activity or function such as having antigenicity, or a base sequence corresponding to the part encodes a part which is specific to the HS2ST and can be used as a primer or probe. means.

The DNA of the present invention also includes DNA or RNA complementary to the DNA of the present invention. Furthermore, the DNA of the present invention may be a single strand of only the coding strand encoding heparan sulfate 2-O-sulfotransferase, and the single strand and a DNA strand or RNA having a sequence complementary thereto. It may be a double-stranded chain.

[0027] The DNA of the present invention is a heparan sulfate 2-
It may have the full length nucleotide sequence of the coding region encoding the entire O-sulfotransferase polypeptide, or may have the nucleotide sequence encoding a part of the heparan sulfate 2-O-sulfotransferase polypeptide It may be something.

In particular, glycosaminoglycan sulfotransferase containing a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 was purified from cultured cells (CHO cells: ATCC CCL61) derived from Chinese hamster ovary by the present inventors. Heparan sulfate 2-O-sulfotransferase (Kobay
ashi, M., Habuchi, H., Habuchi, O., Saito, M., and
Kimata, K. (1996) J. Biol. Chem. 271, 7645-7653) and has the following physicochemical properties. Action: A sulfate group is selectively transferred from a sulfate group donor to a hydroxyl group at the 2-position of an L-iduronic acid residue contained in glycosaminoglycan which is a sulfate group acceptor. That is, a sulfate group is not substantially transferred to the sulfate group acceptor except for the 2-position hydroxyl group of L-iduronic acid. As the sulfate group donor, active sulfuric acid (3′-phosphoadenosine 5′-phosphosulfate; hereinafter also referred to as “PAPS”) is preferably exemplified. The sulfate group is not substantially transferred to the glucosamine residue. Substrate specificity: transfers sulfate groups to heparan sulfate and CDSNS-heparin, but does not transfer sulfate groups to chondroitin, chondroitin sulfate, dermatan sulfate and keratan sulfate. Optimal reaction pH: This enzyme is in the range of pH 5.0 to 6.5,
In particular, it has a high sulfate group transfer activity in a reaction solution having a pH of about 5.5. Optimum reaction ionic strength: The activity of this enzyme increases with an increase in the reaction ionic strength.
It shows the highest activity around 00 mM, especially around 100 mM.
When the NaCl concentration increases beyond this range, the activity gradually decreases, and at 500 mM, the activity becomes extremely low. Inhibition and Activation The activity of this enzyme is increased by coexisting protamine in the reaction solution. With about 0.013 mg / ml or more of protamine, the enzyme activity is increased about 3-fold as compared to the absence of protamine.

The activity of this enzyme is adenosine-3 ',
Inhibition is caused by coexistence of 5'-diphosphate (3 ', 5'-ADP) in the reaction solution. The activity of this enzyme is 1
The co-presence of 0 mM or less of dithiothreitol (DTT) in the reaction solution has almost no effect. Molecular weight: N-glycosidase (Genzym
e) Molecular weight estimated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions after treatment: about 3
8,000.

Molecular weight estimated by superose 12 (Pharmacia-LKB) gel chromatography:
About 130,000. Michaelis constant When CDSNS-heparin is used as a sulfate group acceptor and PAPS is used as a sulfate group donor, the Michaelis constant (Km) of the present enzyme with respect to PAPS is about 0.
20 μM.

A DNA having a nucleotide sequence encoding a sulfotransferase polypeptide having such properties is also included in the DNA of the present invention. Hereinafter, the present invention DN
A method for obtaining A will be described. According to the invention, the invention D
Since the amino acid sequence encoded by the nucleotide sequence of NA has been identified, the DNA of the present invention is obtained from chromosomal DNA or mRNA by PCR (polymerase chain reaction) using oligonucleotide primers prepared based on the sequence. Can also be obtained by amplifying
It can also be produced by cDNA cloning comprising the following steps. (1) The amino acid sequence of at least a part of the purified polypeptide of heparan sulfate 2-O-sulfotransferase is determined. (2) An oligonucleotide primer is prepared based on the amino acid sequence. (3) A probe for the sulfotransferase is produced by amplifying cDNA from RNA extracted from cultured cells by PCR using the above primers. (4) A cDNA library derived from a cultured cell or a living tissue is screened using the probe. By screening, usually, the full-length cDNA of the sulfotransferase is selected.

However, the method for producing the DNA of the present invention is not limited thereto, and the DNA of the present invention can be prepared by the above-mentioned PCR method or other known cDNA cloning techniques.
Can be manufactured.

Hereinafter, an example of the method for producing the DNA of the present invention will be specifically described. (1) Heparan sulfate 2-O-sulfotransferase (HS2S
T) Determination of Amino Acid Sequence (i) Purification of HS2ST Heparan sulfate 2-O-sulfotransferase can be used for heparan sulfate 2-O-sulfotransferase such as cultured cells derived from Chinese hamster ovary.
It is possible to purify cells expressing O-sulfotransferase by combining a normal protein purification method and a normal glycosaminoglycan sulfate transferase purification method. Specifically, J. Biol. Chem.
271, (13), 7645-7653, (1996).

(Ii) Determination of partial amino acid sequence of heparan sulfate 2-O-sulfotransferase It is known that sugar chains are bound to purified HS2ST. Purified HS2S
T is digested with a sugar chain-degrading enzyme such as N-glycanase, and the deglycosylated HS2ST is subjected to SDS-PAGE (S
DS-polyacrylamide gel electrophoresis)
Polyvinylidene fluoride (polyvinylidene fluoride;
(PVDF) film or nitrocellulose film. This membrane is stained with a dye that stains proteins such as Coomassie Brilliant Blue (CBB) and Amido Black,
The protein band formed after N-glycanase digestion is cut out and used for fragmentation.

The method of fragmentation is not particularly limited, but the protein can be fragmented by a known method such as by contacting the protein band with a protease. Specific examples of proteolytic enzymes include endoproteinase Lys-C and endoproteinase Asp.
-N and the like. The band was cut out of the gel and contacted with a protease, followed by SDS-PAGE
For example, they may be separated. As a simple operation, Clevel a
nd, DW, Fischer, SG, Kirshner, MW, and La
emmli, UK (1977) J. Biol. Chem. 252, 1102-1106
There is a method. That is, a protein band is cut out and inserted into a well of another gel, a buffer containing a proteolytic enzyme is placed on the inserted gel, and SDS-PAGE is performed. In this method, electrophoresis is suspended by temporarily cutting off the enzyme, enzymatic digestion is performed for about 30 minutes, and then electrophoresis is restarted. This method is preferable because enzymatic digestion and separation of the peptide fragment after digestion can be performed in a single step. After transferring the peptide generated by fragmentation to a PVDF membrane or a nitrocellulose membrane, the peptide is stained with CBB or amide black, and the peptide band is cut out. The amino-terminal sequence of a peptide can be determined by a known method on a PVDF membrane or a nitrocellulose membrane containing a peptide formed after digestion with a proteolytic enzyme. Specifically, a model 476A protein sequencer (Applied Biosystems)
s) (manufactured by S. Co., Ltd.), but the present invention is not limited thereto. In addition, it is also possible to ask a vendor to determine the amino acid sequence.

(Iii) Synthesis of oligonucleotide primers Based on the partial amino acid sequence of HS2ST, oligonucleotide primers for PCR are prepared. It is preferable to use a site in the amino acid sequence where codon degeneracy is as small as possible. Examples of such primers are shown in FIG. 1 (sense primers: SEQ ID Nos. 8, 9; antisense primers: SEQ ID Nos. 10, 11).

(2) Preparation of HS2ST partial cDNA and preparation of probe Total RNA was prepared by a known method (Kingston, RS, (1991)).
in Current Protocols in Molecular Biology, Suppl.
14, Unit 4.2, Greene Publishing Associates and Wil
ey Interscience, New York, etc.). The material is m of heparan sulfate 2-O-sulfotransferase.
The material is not limited as long as it is a material expressing RNA,
Cultured cells are preferred in terms of ease of handling and proliferation. Among cultured cells, Chinese hamster ovary cells (CHO cells: ATCC CCL61) are particularly preferred because the enzyme is strongly expressed and the enzyme activity is relatively high. The medium used for culturing the cultured cells is not particularly limited, but a medium suitable for culturing suspended cells using a spinner flask or the like is preferable for efficiently obtaining a large amount of cells. Specifically, CH
When O cells are used, CHO-SS for suspension culture
A commercially available medium such as an FMII medium (manufactured by Gibco) may be used. What is necessary is just to culture | cultivate similarly to a normal culture cell using a spinner flask using the above-mentioned culture media. The cultivation is preferably performed in a carbon dioxide gas incubator, and it is preferable to adjust the concentration of carbon dioxide in the incubator to 5 to 7% and the air to 95 to 93%. Further, the temperature is preferably adjusted to about 37 to 38 ° C.

[0038] Total RNA can be obtained from the cultured cells cultured as described above by a commonly used method for preparing total RNA. The method is based on the guanidine thiocyanate / CsCl method.
(Kingston, RE, (1991) in Current Protocols in M
olecular Biology, Suppl. 14, Unit 4.2, Greene Publ
ishing Associates and Wiley Interscience, New Yor
It is preferably prepared in k).

The poly (A) ⁺ RNA prepared poly (A) ⁺ RNA, total RNA obtained as described above
Can be purified by, for example, oligo- (dT) cellulose column chromatography.

HS2ST partial cDN by PCR method
A. Amplification of HS2ST partial cDNA can be performed by reverse transcription PCR using the above poly (A) ⁺ RNA as a template and oligonucleotide primers. The PCR may be carried out in the same manner as in a usual method, but a specific method is as follows. 1 μl of poly (A) ⁺ RNA, 100
pmol of the above-mentioned oligonucleotide primer, 500 μM of each of the four deoxynucleoside triphosphates, 200 units of M-MLV reverse transcriptase (Gibco BRL)
(Gibco BRL)), 1 mM dithiothreitol (DT
T) a buffer solution containing 120 units of RNase (ribonuclease) inhibitor (manufactured by Takara Shuzo Co., Ltd.) (final volume 2)
0 μl) was incubated at 37 ° C. for 60 minutes, cDN
Synthesize A primary strand. Next, the above reverse transcription reaction mixture 2
A reaction solution (final volume: 50 μl) containing μl, 1 μM oligonucleotide primers, 200 μM each of four kinds of deoxynucleoside triphosphates, and 1.25 units of Taq polymerase was added at 94 ° C. for 1 minute, at 60 ° C. for 2 minutes,
The process is repeated at 72 ° C. for 2 minutes for 3 cycles, then the temperature of the process at 60 ° C. for 2 minutes is reduced to 2 ° C. every 3 cycles to 48 ° C., and further repeated at 48 ° C. for 17 cycles.

The partial cDNA thus obtained
Is used as a hybridization probe for screening a full-length cDNA (cDNA containing the entire coding region) from a cDNA library.

(3) Preparation of cDNA Library (i) Synthesis of cDNA and Preparation of Recombinant DNA cDNA should be synthesized by a normal method using a reverse transcriptase reaction using poly (A) ⁺ RNA as a template. Can be. For synthesis, it is convenient to use a commercially available cDNA synthesis kit. For example, TimeSaver cDNA syn
Thetheskit (Pharmacia LKB Biotechnology) can also be used to synthesize cDNA and ligate the cDNA to a cloning vector. Also,
By using a commercially available cDNA library, cDNA can be obtained more easily. In the present invention, a Lamda ZAP library manufactured by Stratagene, which is a cDNA library of CHO cells, and a λgt11 library manufactured by Clontech, which is a cDNA library derived from human fetal brain, are used. These recombinant DNAs linked to a cloning vector are introduced (transfected) into host bacterial cells. The host bacterial cell to be used must be selected depending on the cloning vector to be used, but usually E. coli (Escherichia
Escherichia coli (Escherichia coli) is often used in combination with a cloning vector using Escherichia coli (E. coli) as a host, but is not limited thereto. Transfection is usually performed by mixing the recombinant DNA with E. coli having altered cell membrane permeability in the presence of 30 mM calcium chloride. In the case of a λ phage vector such as Lamda ZAP or λgt11, the recombinant DNA can be directly introduced into E. coli which has been treated with calcium chloride. An efficient infection method is generally used, and packaging can be performed using a commercially available packaging kit (Gigapack II packaging extract, manufactured by Stratagene, etc.). The packaged recombinant DNA is
E. coli is transfected, and it is necessary to select an E. coli strain to be used depending on the cloning vector used. That is, when a cloning vector containing an antibiotic resistance gene is used, Escherichia coli must have no resistance to the antibiotic, and when a cloning vector containing a gene such as the β-galactosidase gene (lacZ) is used. It is necessary to select Escherichia coli that does not express β-galactosidase activity. This means
That is what recombinant DNA is needed to screen for transfected E. coli. For example, L
When an amda ZAP or λgt11 cloning vector is used, E. coli is used. coli XL-1 Blue or E. coli. c
An E. coli strain such as oliY1088 may be selected. Escherichia coli into which the recombinant DNA or recombinant plasmid has been introduced can be screened by obtaining resistance to antibiotics, obtaining β-galactosidase activity, and the like.
Specifically, E. coli is spread on an agar medium, and the grown colonies may be selected. The grown E. coli (E. coli transfected with the recombinant DNA) constitutes a cDNA library. When Bluescript is used for the plasmid, it may be suspended in a soft agar medium together with the indicator bacterium and overlaid on an agar medium to form plaque. DN
Phage plaques carrying the plasmid into which the A fragment has been inserted can be easily selected because they do not express β-galactosidase activity.

(Ii) HS2ST full-length cDNA cloning Next, from the cDNA library obtained as described above, a phage clone having the HS2ST full-length cDNA is selected by hybridization using the HS2ST partial cDNA as a probe. it can. Hybridization may be performed according to a usual method. From the selected positive clone, HS2ST is prepared by preparing phage DNA and cleaving it with an appropriate restriction enzyme.
cDNA can be cut out. Obtained cDNA
Is determined as it is or by subcloning into an appropriate plasmid to determine the nucleotide sequence.

The nucleotide sequence of the HS2ST cDNA derived from Chinese hamster determined as described above and the amino acid sequence deduced from this nucleotide sequence are shown in SEQ ID NO: 1.
SEQ ID NO: 2 shows only the amino acid sequence. In addition, the nucleotide sequence of human-derived HS2ST cDNA and the amino acid sequence predicted from this nucleotide sequence are shown in SEQ ID NO: 3, and only the amino acid sequence is shown in SEQ ID NO: 4. HS2STc of human origin
The DNA is HS2S derived from the Chinese hamster described above.
It can also be obtained by screening a human-derived cDNA library using T as a probe.

<2> A polypeptide comprising all or a part of a glycosaminoglycan sulfotransferase polypeptide encoded by the nucleotide sequence of the DNA of the present invention. Also provided is a polypeptide comprising all or a portion of a noglycan sulfotransferase polypeptide. In the present specification, the above “portion” refers to a portion having some activity or function such as having HS2ST activity or having antigenicity. The present polypeptide may be used alone or may be fused with another polypeptide. The polypeptide may not have a sugar chain.

HS2ST expressed in vivo in mammals
Has a sugar chain, and thus is clearly distinguished from the present polypeptide having no sugar chain. Such a polypeptide can be obtained by the method for producing a polypeptide described below.
For example, by introducing the present invention into mammalian cells,
A polypeptide in which a sugar chain is added to the present polypeptide can be produced, and only a polypeptide having no sugar chain can be produced by introducing the polypeptide into a prokaryotic cell such as Escherichia coli. The determination of the presence or absence of the above activity or function can be performed by a method known to those skilled in the art.

In particular, HS2ST containing a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 is a novel heparan sulfate 2-O-sulfotransferase expressed in human tissues. It has an amino acid sequence represented by No. 4 and substantially has an enzyme activity of transferring a sulfate group from a sulfate group donor to a hydroxyl group at the 2-position of an L-iduronic acid residue contained in glycosaminoglycan which is a sulfate group acceptor. Provided is a glycosaminoglycan sulfotransferase comprising a polypeptide which may have one or more amino acid residue substitutions, deletions, insertions, or rearrangements that do not harm the environment.

<3> HS2ST using DNA of the present invention
A method for producing a polypeptide The cells transformed with the DNA of the present invention are cultured in a suitable medium, and the polypeptide encoded by the DNA of the present invention is produced and accumulated in the culture. By collecting, a polypeptide of HS2ST can be produced.

The cells transformed with the DNA of the present invention can be obtained by inserting a fragment of the DNA of the present invention into a known expression vector, constructing a recombinant plasmid, and performing transformation using the recombinant plasmid. Can be. Examples of the cells include prokaryotic cells such as Escherichia coli and eukaryotic cells such as mammalian cells.

In this production method, a host-vector system usually used for production of proteins can be used. For example, mammalian cells such as COS-7 cells and pCXN
2 (Niwa, H., Yamanura, K. and Miyazaki, J. (1991)
It is preferable to employ a combination of mammalian cell expression vectors such as Gene 108, 193-200) or pFLAG (Eastman Kodak). The culture medium and culture conditions are appropriately selected according to the host used, that is, the cells.

The DNA of the present invention may be directly expressed,
It may be expressed as a fusion polypeptide with another polypeptide. Further, the DNA of the present invention may be expressed in full length, or may be partially expressed as a partial peptide.

The polypeptide of the present invention can be collected from the culture by a known polypeptide purification method. The culture includes a medium and cells in the medium.

[0053]

EXAMPLES The present invention will be described more specifically with reference to the following examples. <1> Chinese hamster heparan sulfate 2-O-
Preparation of Sulfotransferase and Analysis of Amino Acid Sequence As a fraction eluted from the second 3 ′, 5′-ADP-agarose column by the method described in J. Biol. Chem. 271, 7645-7653 (1996). Purified HS2ST was obtained. S
For DS-polyacrylamide gel electrophoresis (PAGE), 28 μg of HS2ST was precipitated with 10% trichloroacetic acid and washed twice with acetone. This precipitate was reduced with a loading buffer containing 5% (V / V) 2-mercaptoethanol at 100 ° C. for 3 minutes and subjected to SDS, and then subjected to Laemmli (Laemmli, UK (1970) Na).
227, 680-685), and SDS-PAGE was performed using a 10% polyacrylamide gel. S
ProBlott PVDF membrane (Applied Biosystems) at 200 mA for 2.5 hours in a 10 mM 3-cyclohexylamino-1-propanesulfonic acid (CAPS) solution containing 10% methanol at pH 11 Transfer). The transcribed protein was obtained using the method of Aebersold et al.
ld, RH, Leavitt, J., Saavedra, RA, Hood, LE,
and Kent, SBH (1987) Proc. Natl. Acad. Sci. USA
84, 6970-6974). 45k
The band in the region below Da was cut out and subjected to the Iwamatsu method (Iwamatsu, A. (1992) Electrophoresis 13, 142-147).
Was modified on a PVDF membrane and S-carboxymethylated by a modified method. The protein transferred to the membrane
0.5 M Tris-HCl, pH 8.8, 5% (V /
V) Acetonitrile, 1 mg dithiothreitol (D
It was reduced in 300 μl of an 8 M guanidine hydrochloride solution containing TT) for 1 hour at room temperature. Thereto was added 12 μl of a 1N NaOH solution containing 3 mg of iodoacetic acid, and the mixture was placed in a dark place for 15 minutes. The membrane is washed with distilled water and then 0.1%
Washed with 2% acetonitrile containing SDS. The S-carboxymethylated protein on the membrane was 0.5% dissolved in 100 mM acetic acid containing 1 mg methionine.
Polyvinyl pyrrolidine (PVP-40) 300 μl
Incubate at room temperature for 30 minutes in 10% (V /
V) Washed with acetonitrile. Cut the membrane finely,
The cells were treated with 0.3 U of N-glycanase dissolved in 50 μl of Tris-HCl (pH 7.5) for 15 hours at 37 ° C. Thereafter, in situ sequential digestion was performed at 10% (V
/ V) 20 mM at pH 9.0 with acetonitrile
Endoproteinase L dissolved in Tris-HCl such that enzyme: substrate (mol: mol) is 1:50
15 h at 37 ° C. with ys-C, followed by 10%
(V / V) 20m of pH 7.5 containing acetonitrile
Enzyme: substrate (mol: mo) in a reaction solution of pH 7.8 containing M ammonium bicarbonate and 25 mM CaCl2.
l) was performed at 40 ° C. for 24 hours with endoproteinase Asp-N dissolved at a ratio of 1:50. The digest was collected, filtered and lyophilized. Mobile phase A containing 1% (V / V) acetonitrile (0.06% (V / V) trifluoroacetic acid (TF
A)) This lyophilized product was dissolved in 18 µl, and subjected to capillary HPLC on a reversed-phase column (0.3 x 150 mm). The peptide was eluted at a flow rate of 3.3 μl / min, 100
2% to 100% mobile phase B (0.052%
(80% acetonitrile containing TFA). Peptide fractions were manually collected while monitoring the absorbance at 214 nm and blotted onto small pieces of PVDF membrane. Amino acid sequencing was performed using a model 476A protein sequencer (Applied Biosystems (Appl.
ied Biosystems)). Table 1 shows the results.

[0054]

[Table 1] ──────────────────────────────────── peptide number amino acid sequence sequence number ──── ──────────────────────────────── 1 DLCAKNRYHVLHI 5 2 DQVRFVKNI 6 3 DXYRPGLXR 74 4 DIVIXYNR 85 DLYR 9 ─── ─────────────────────────────────

<2> Amplification of HS2ST partial cDNA by PCR (1) Preparation of PCR primer Based on the above peptides 1 and 2, degenerate oligonucleotides of terminal and internal primers having deoxyinosine substitution shown in FIG. 1 (Primers 1s (SEQ ID NO: 10) and 1si (SEQ ID NO: 11) having a template DNA sequence, primers 2a (SEQ ID NO: 12) and 2ai (SEQ ID NO: 13) having a complementary sequence to the template).

(2) PCR reaction A polyclonal sample collected from CHO cells by an ordinary method using oligo- (dT) cellulose chromatography.
(A) Using ⁺ RNA as a template for a reverse transcription reaction and oligo dT as a primer to synthesize a single strand of cDNA,
Used as template for R. PCR was performed using a mixture of 1 μM terminal primers 1s and 2a (or 1si and 2ai), 2
μl of reverse transcription reaction, 200 μM of each of the four deoxynucleotide triphosphates, and 1.25 U of Am
pliTaq polymerase (Perkin-Elmer (Perki)
n-Elmer). Amplification was performed as follows. In the first three cycles, the dissociation reaction was performed at 94 ° C. for 1 minute, annealing was performed at 60 ° C. for 2 minutes, and the extension reaction was performed at 72 ° C. for 2 minutes, and only the annealing temperature was reduced by 2 ° C. to 50 ° C. every three cycles. Finally, 17 cycles were performed at the annealing temperature of 48 ° C. Thereafter, an extension reaction was performed for another 15 minutes. When the amplified substance generated by this operation was analyzed by agarose gel electrophoresis, an amplified DNA band of about 90 bp was detected. Even when PCR was performed using primers 1si and 2si, which are shifted 9 bp and 3 bp respectively from the 3 ′ end with respect to 1s and 2a, DNA fragments of almost the same size were generated.

<3> HS2S of Chinese Hamster
Acquisition of T full-length cDNA (1) Preparation of hybridization probe The DNA fragment obtained by performing PCR using 1s and 2a as primers is obtained from Jetsorb (Genomed).
Was used to recover. This DNA, which had been blunted using T4 DNA polymerase and phosphorylated with T4 polynucleotide kinase, was ligated to an EcoRV digested fragment of alkaline phosphatase-treated Bluescript plasmid (manufactured by Stratagene) DNA, and JM109 was ligated. And subcloned by selection with blue and white colors. Subclones were confirmed by sequencing.

The radiolabeled probe used for the screening of the cDNA library was obtained by using the primer 1s
And 2a, about 90b subcloned as template
p, and [α- ³² P] dCTP (Amersham (A
Ps amplified in a final volume of 25 μl of a solution containing
Prepared from CR product. PCR was performed at 94 ° C. for 1 minute, 48
1 minute at 72 ° C. and 1 minute at 72 ° C. were repeated 35 times,
The final cycle was performed by extending the extension time at 72 ° C. for another 15 minutes.

(2) Screening of HS2ST cDNA clone
Lamda, a cDNA library for leaning CHO cells
Purchase ZAP cDNA library from Stratagene
Entered. E. coli host coli XL-1 Blue cells
Phages of the library were infected. One plate per plate
2-4 × 10 ^FourSo that plaques are formed
About 1.4 × 10⁶Screening colonies
Was. Rolls generated from Uni-ZAP XR library
Hybond N + nylon membrane with knee
Fixed by the re-fixation method, 37.5% formamide, 5 ×
SSPE (sodium chloride / sodium phosphate / EDT
A buffer), 5 × Denhard's solution, 0.5% SD
Contains S and 50 μg / ml denatured salmon sperm DNA
Prehybridization was performed in the solution at 45 ° C for 3.5 hours.
³²The P-labeled probe was added to the above buffer, and 4
Hybridized at 2 ° C. for 16 hours. Filter 5
1 × SSPE, 1% SDS, and 0.1 × S at 5 ° C.
Wash with SPE, 0.1% SDS, autoradio
Six positive clones were detected by chromatography.

(3) HS derived from Chinese hamster
Nucleotide sequence of 2ST cDNA Bluescript plasmid from a positive clone was
ExAssist helper phage and E. coli coli S
Stratagene in vivo DN using OLR
A extraction method (Stratagene in vivo excision protocol)
And cut out. Bluescript plasmid DNA introduced into SOLR was purified using QIAGEN plasmid kit. Among the introduced cDNAs, the nucleotide sequences of the longest cDNAs named K3 and H8 of 2.2 kbp were determined. The base sequence is dGTP / deazaGT
It was confirmed using the P kit and Sequenase version 2.0 (each manufactured by US Biochemical). DNA synthesis was started with T3 DNA polymerase and T7 DNA polymerase, and an internal primer was inserted at a position of about 250 bp. The obtained DNA is used as the computer software Genetics-Mac (GENE
(TYX-MAC: manufactured by Software Development Co., Ltd.). As a result, it was revealed that the cDNA obtained from K3 contained the entire sequence obtained from H8 and contained the entire region encoding HS2ST, and the amino acid sequence encoded from this sequence was predicted (SEQ ID NO: 1). . The amino terminal sequence contained four in-frame ATG codons. There was a TGA sequence for the stop codon at the location -21 upstream of the first ATG codon. The open reading frame starting from the first ATG codon is 356
A protein having 41,830 Da of amino acid residues and two sugar bondable regions was expected. The hydropathy plot of this amino acid sequence revealed that 14 amino acid residues from the 14th to the 27th in the HS2ST amino-terminal region were clear hydrophobic regions (FIG. 2). The amino acid sequence predicted to be a base is compared with the base sequence of DNA encoding another protein and the protein database (EMBL-GDB
Release 44 and NBRF-PDB Release 45). As a result, homology to other known sulfotransferases was recognized except that the DLIYL sequence present at amino acids 179 to 183 of chicken chondroitin 6-sulfotransferase was conserved at amino acids 338 to 342. I couldn't. In addition, it has been reported that allylsulfotransferase IV, which is conserved with a relatively high probability, exhibits affinity with PAPS for sulfotransferases GXXGXXK or LE.
KCGR sequence (Zheng, Y., Bergold, A., and Duffel,
MW (1994) J. Biol. Chem. 269, 30313-30319) could not be found. From the amino acid sequence, after treating the purified protein with endoproteinase Asp-N, the amino acid sequence of the fragment obtained by treating with endoproteinase Lys-C was found, and this cDNA clone encodes purified HS2ST. It was decided to do.

<4> Acquisition of Human HS2ST Full-Length cDNA (1) Screening of HS2ST cDNA Clone Using the Chinese hamster HS2ST cDNA as a screening probe, human-derived H
The S2ST full-length cDNA was screened. Λgt1 incorporating a human fetal brain cDNA library
1 cDNA library was purchased from Clontech. E. coli host E. coli Y1088 cells were infected with the phage of the library. 2 to 4 per plate
Spread so that × 10 ⁴ plaques are formed.
0 × 10 ⁶ colonies were screened. λgt
Hybo to which a colony generated by incorporating No. 11 was transcribed
fix the ndN + nylon membrane by the alkali fixation method,
7.5% formamide, 5 × SSPE (sodium chloride / sodium phosphate / EDTA buffer), 5 × Denhard '
prehybridization at 42 ° C. for 3.5 hours in a solution containing 0.5% SDS and 50 μg / ml denatured salmon sperm DNA. ^{The 32} P-labeled probe was added to the above buffer, and hybridized at 42 ° C. for 16 hours. Filter at 45 ° C. with 1 × SSPE, 1%
SDS, further 0.1 × SSPE, 0.1% SDS
And seven positive clones were detected by autoradiography.

(2) Base sequence of HS2ST cDNA Bluescript plasmid from a positive clone was
ExAssist helper phage and E. coli coli S
Stratagene in vivo DN using OLR
A extraction method (Stratagene in vivo excision protocol)
And cut out. The Bluescript plasmid DNA introduced into SOLR was purified using a QIAGEN plasmid kit, and the nucleotide sequence of the cDNA was determined. The base sequence is the Sequs enclosed with the dGTP / deaza GTP kit.
enase version 2.0 (each US Biochemical
(Biochemical)). DNA synthesis was started with T3 DNA polymerase and T7 DNA polymerase. The obtained DNA was edited and analyzed by computer software Genetics-Mac (GENETYX-MAC: manufactured by Software Development). As a result, the nucleotide sequence of the entire region encoding human HS2ST was clarified, and the amino acid sequence encoded from this sequence was predicted (SEQ ID NO: 3). The amino terminal sequence contained four in-frame ATG codons. A stop codon T at the site upstream-21 of the first ATG codon
GA sequence was present. The open reading frame starting from the first ATG codon was predicted to be a protein having two sugar binding possible regions at 41868 Da of 356 amino acid residues. The hydropathy plot of this amino acid sequence revealed that 14 amino acid residues from the 14th to the 27th in the HS2ST amino-terminal region were clear hydrophobic regions. The amino acid sequence predicted as a base was compared with the above-mentioned cDNA derived from Chinese hamster and HS2ST encoded thereby. As a result, HS2ST derived from human is HS2S derived from Chinese hamster.
It was found to have 97.5% homology with T (FIG. 3).

<5> Expression of HS2ST cDNA (1) Construction of HS2ST Expression Plasmid In order to express HS2ST cDNA, a cDNA fragment was inserted into an expression vector to construct a recombinant plasmid. The isolated cDNA was transformed into a mammalian expression vector pcD.
PcDNA3, a recombinant plasmid introduced into NA3
HS2ST was constructed.

(2) HS2STc in COS-7 cells
Transient (Transient) Expression of DNA COS-7 cells were used as a host for HS2ST cDNA expression.

The cells transfected with pcDNA3HS2ST were cultured for 67 hours.
M., Habuchi, H., Habuchi, O., Saito, M., and Kima
Cell extract was prepared according to the method described in ta, K. (1996) J. Biol. Chem. 271, 7645-7653. After centrifugation of this cell extract at 10,000 xg for 30 minutes at 4 ° C, HS2ST, HS6ST, chondroitin O-
Sulfotransferase (COST) activity was examined. As controls, COS-7 cells transfected with pcDNA3 without cDNA and COS with no transfection
-7 cells were used. When transfected with a vector containing isolated Chinese hamster-derived cDNA, the HS2ST activity was increased 2.6-fold over the control without any transfections (Table 2). These activities were measured by Kobayashi, M., Habuchi, H., Habuchi, O.,
Saito, M., and Kimata, K. (1996) J. Biol. Chem. 2
71, 7645-7653.

[0066]

[Table 2] 硫酸 Sulfotransferase activity───── ──────────────────────── HS2ST HS6ST COST ───────────────────────対 照 Control 2.2 ± 0.5 1.7 ± 0.5 6.4 ± 0.1 pcDNA3 2.2 ± 0.1 1.9 ± 0.3 6.7 ± 0.5 pcDNA3HS2ST 5.7 ± 0.7 1.6 ± 0.4 7.0 ± 0.1 ─────── ───────────────────────────── * The unit of the numerical value in the table is pmol / min / mg protein

As shown in the table, the cD isolated above
HS2ST of cells carrying a vector expressing NA
The activity was about 2.5 times that of the control and the cells transfected with the plasmid not carrying the cDNA. HS6ST
No increase in activity or COST activity occurred. From these results, it was proved that the isolated cDNA encoded a protein having HS2ST activity. In addition, human HS2ST was expressed by transfecting COS-7 cells using the human HS2ST cDNA in the same manner as described above. As a result, a result similar to that obtained when HS2ST of Chinese hamster was expressed was obtained, and the cDNA of HS2ST derived from human had an activity similar to that of HS2ST derived from Chinese hamster.
It turned out that it codes S2ST.

[0068] <6> Chinese hamster ovary cells poly (A) ⁺ RNA poly extracted from the analysis Chinese hamster ovary cell line CHO of HS2ST expression by Northern blot (A) ⁺ 50% of the RNA to pH7.0
Formamide (V / V), 6% formaldehyde (V
/ V), 20 mM MOPS buffer at 65 ° C, 10
After denaturation for 2 minutes, electrophoresis was performed on a 1.2% agarose gel containing 6% formaldehyde (V / V). 50 mM
NaOH for 20 minutes, neutralize with 20 × SSC (sodium acetate / sodium chloride buffer) for 45 minutes, transfer the RNA in the gel to Hybond N ⁺ nylon membrane overnight, and use 50 mM NaOH for 5 minutes. Fixed. The RNA immobilized on the membrane was treated with 50% formaldehyde, 5 × SSPE, 5 × Denhardt's solution, 42 × C at 42 ° C. for 3 hours.
Prehybridization was performed in a solution containing 5% SDS, 100 μg / ml denatured salmon sperm DNA. Hybridization was performed with the above buffer containing a ³² P-labeled probe (1 × 10 ⁶ cpm / ml). The radiolabeled probe was a D3 consisting of 1,145 bases between base numbers 113 to 257 in SEQ ID NO: 1 obtained by digesting the K3 clone inserted into Bluescript with SmaI and AflII.
[Α- ³² P] dCTP and Ready-To-G
o Using a DNA labeling kit (Pharmacia Biotech), random oligonucleotide-prime labeling
tide-primed labeling) method. This film is 6
After washing with 1 × SSPE and 0.1% SDS at 5 ° C., the substrate was washed with 0.1 × SSPE and 0.1% SDS at the same temperature. This film was exposed to an X-ray film using a sensitizing film at -80 ° C for 14 hours. As a result, 5.0 kb
And two bands of 3.0 kb were obtained.

[0069]

Industrial Applicability According to the present invention, heparan sulfate 2-O-sulfotransferase (HS2) which selectively transfers a sulfate group to a hydroxyl group at the 2-position of L-iduronic acid residue contained in heparan sulfate.
ST) and a DNA having a base sequence encoding the polypeptide are obtained. Further, DN derived from the DNA
A polypeptide expressed from the A fragment is obtained.

According to the present invention, a DNA having a nucleotide sequence encoding an HS2ST polypeptide was obtained.
It is expected that HS2ST can be mass-produced to the extent that it can be used industrially.

[0071]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 2138 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: cDNA Origin Organism name: Chinese hamster Tissue type: Ovary Sequence characteristics Characteristic symbols : CDS Location: 24..1091 Method for determining characteristics: Features of P sequence Symbol indicating characteristics: transmembrane domain Location: 63..104 Method for determining characteristics: Characteristics of P sequence Symbol for indicating characteristics: potential N -glycosylation site Location: 345..353 Method for determining characteristics: Features of S sequence Symbol indicating characteristic: potential N-glycosylation site Location: 403..410 Method for determining characteristics: S sequence CTTGATCTCC AGCCGCGGGT TTC ATG GGG CTC CTC AGG ATC ATG ATG CCG 50 Met Gly Leu Leu Arg Ile Met Met Pro 15 5 CCC AAG TTG CAG CTG CTG GCG GTG GTG GCC TTC GCC GTG GCG ATG CTC 98 Pro Lys Leu Gln Leu Leu Ala Val Val Ala Phe Ala Val Ala Met Leu 10 15 20 25 TTC TTG GAG AAC CAG ATC CAG AAG CTG GAG GAG TCC CGG GCG AAG CTA 146 Phe Leu Glu Asn Gln Ile Gln Lys Leu Glu Glu Ser Arg Ala Lys Leu 30 35 40 GAA AGG GCA ATC GCA AGA CAT GAA GTC CGG GAA ATT GAA CAG CGG CAT 194 Glu Arg Ala Ile Ala Arg His Glu Val Arg Glu Ile Glu Gln Arg His 45 50 55 ACA ATG GAT GGC CCT CGG CAA GAT GCG GCT GTA GAT GAA GAA GAA GAT 242 Thr Met Asp Gly Pro Arg Gln Asp Ala Ala Val Asp Glu Glu Glu Asp 60 65 70 ATA GTC ATC ATT TAT AAC AGA GTT CCC AAA ACT GCA AGC ACC TCG TTT 290 Ile Val Ile Ile Tyr Asn Arg Val Pro Lys Thr Ala Ser Thr Ser Phe 75 80 85 ACC AAT ATC GCC TAT GAC TTG TGT GCG AAG AAT AGA TAC CAT GTT CTT 338 Thr Asn Ile Ala Tyr Asp Leu Cys Ala Lys Asn Arg Tyr His Val Leu 90 95 100 105 CAC ATC AAC ACT ACC AAA AAC AAC CCA GTG ATG TCA TTG CAA GAT CAG 386 His Ile Asn Thr Thr Lys Asn Asn Pro Val Met Ser Leu Gln Asp Gln 110 115 120 GTA CGC TTT GTA AAG AAT ATA ACC ACT TGG AAC GAG ATG AAA CCA GGG 434 Val Arg Phe Val Lys Asn Ile Thr Thr Trp Asn Glu Met Lys Pro Gly 125 130 135 TTT TAT CAT GGA CAC ATT TCT TAT CTG GAT TTT GCA AAA TTC GGT GT G 482 Phe Tyr His Gly His Ile Ser Tyr Leu Asp Phe Ala Lys Phe Gly Val 140 145 150 AAG AAG AAG CCC ATT TAC ATT AAT GTC ATC AGG GAC CCT ATC GAG AGG 530 Lys Lys Lys Pro Ile Tyr Ile Asn Val Ile Arg Asp Pro Ile Glu Arg 155 160 165 CTT GTT TCC TAC TAT TAC TTT CTG AGG TTT GGG GAT GAT TAC AGA CCA 578 Leu Val Ser Tyr Tyr Tyr Phe Leu Arg Phe Gly Asp Asp Tyr Arg Pro 170 175 180 185 GGA TTA AGG AGA CGG AAA CAA GGA GAC AAA AAG ACC TTT GAT GAA TGT 626 Gly Leu Arg Arg Arg Lys Gln Gly Asp Lys Lys Thr Phe Asp Glu Cys 190 195 200 GTG GCT GAG GGC GGC TCA GAC TGT GCT CCG GAG AAG CTC TGG CTC CAG 674 Val Ala Glu Gly Gly Ser Asp Cys Ala Pro Glu Lys Leu Trp Leu Gln 205 210 215 ATC CCA TTT TTC TGT GGC CAC AGC TCA GAA TGC TGG AAT GTG GGA AGC 722 Ile Pro Phe Phe Phe Cys Gly His Ser Ser Glu Cys Trp Asn Val Gly Ser 220 225 230 AGA TGG GCT ATG GAT CAA GCT AAG TAT AAC CTC ATT AAC GAG TAC TTT 770 Arg Trp Ala Met Asp Gln Ala Lys Tyr Asn Leu Ile Asn Glu Tyr Phe 235 240 245 CTG GTG GGA GTT ACT GAG GAG CTG GAA GAC TTC ATC AT G CTA CTC GAG 818 Leu Val Gly Val Thr Glu Glu Leu Glu Asp Phe Ile Met Leu Leu Glu 250 255 260 265 GCA GCT TTG CCC CGG TTT TTC CGG GGT GCT ACA GAC CTC TAT CGT ACA 866 Ala Ala Leu Pro Arg Phe Phe Arg Gly Ala Thr Asp Leu Tyr Arg Thr 270 275 280 GGA AAG AAA TCC CAC CTG AGG AAA ACC ACA GAG AAG AAA CTT CCC ACC 914 Gly Lys Lys Ser His Leu Arg Lys Thr Thr Glu Lys Lys Leu Pro Thr 285 290 295 AAG CAA ACC ATC GCG AAG CTG CAG CAG TCT GAC ATT TGG AAA ATG GAA 962 Lys Gln Thr Ile Ala Lys Leu Gln Gln Ser Asp Ile Trp Lys Met Glu 300 305 310 AAT GAG TTC TAC GAG TTT GCA CTA GAG CAG TTC CAG TTC ATC AGA GCC 1010 Asn Glu Phe Tyr Glu Phe Ala Leu Glu Gln Phe Gln Phe Ile Arg Ala 315 320 325 CAC GCT GTC CGT GAG AAA GAT GGA GAC CTC TAC ATC CTG GCC CAG AAC 1058 His Ala Val Arg Glu Lys Asp Gly Asp Leu Tyr Ile Leu Ala Gln Asn 330 335 340 345 TTT TTC TAT GAA AAG ATT TAC CCG AAG TCG AAC TGAGTGGAAG TGTGACCAGA 1111 Phe Phe Tyr Glu Lys Ile Tyr Pro Lys Ser Asn 350 355 GCAGTCTTGA ACCTGGACTT GGCTGTGTTG TCACCGTTGT TCTCAGCTT C TGCACCTGTT 1171 CTGCTAATCG AGTCCAAGCC GAGCCAGTTC TTGTTGGGCC GAGTTGGGGA ACAGACAGGA 1231 GTGTCAAGAA ATTAGATGCT GAATGGGATG TCAGTGTTCT AAGGAGTTCT TAAGTTCTTA 1291 AGTGTGATGA ATTGTTATTT CTTTTGTTAC TTTGTTTTCA TTTTCATGAT AGCTATAATC 1351 TCCCAGTGAG GAGAAATCTC ATGTCACTTA AAATACACAC ATGGAGGTTT AATCAGAAGG 1411 CTGAATACCA TTTCAGAAGA GGTTCTGTGA TTCTCTTGCT TTTGATGAAG CATTTTTATC 1471 ACCTCTCTTT GGATGCAGAT GAGTCTGTAT GGCACTTGGA GTTTTGTGTT GCACACCCCT 1531 ACTGGATAGT GCTAATAACT ATTTGCCAGT AGCTGATTTG TTTATGTGGA TCACGTCTCA 1591 CAGAGTTTAT TGGAATGTTT GATCATGTTT TCTCAGAACT GTTTTTGCTG TAGTTGAGTT 1651 TGCCCATATT TATGTAGGCT TTATTTTATT TTTTGGATGA TCATTAGTGT TAAAGAAATC 1711 AACTGAAAAC CATGAATAAT ACTGTAAAAA GACAAAACAG TTAAAAGCAG TATTCCTGAT 1771 TTCTGTCTCC CCAGTATCTA ATATTGGGGT GGTATTTCTA AGAATGTTGA CAACATTATC 1831 TGAGGCTTTC TTAAGGATTT CCACACATTC ATATAAAAAA AATGAGTTTA GTATTTGTTT 1891 CTCCATGGCT TCTCTATAAC CCAGTACACT GAAGTATCGG TGACTGCATA TGGCAACTCC 1951 ATCAGTGAGC TGTGATGGTA GGATTTTCCT ACCTCTGTAC TTTTACCTGT AGAC TATTTT 2011 TACTACGGTG CTTTATAATG TGTTTTAAAG CATTGCATTT ACAAAAGAAA AATGCTGTAA 2071 ATATTGCATA TTTTATGTAT TTGGACCAAA AAGTTACAAG TCAGTAGATA AAAAGTGGTT 2131 TTGCACC 2138

SEQ ID NO: 2 Sequence length: 356 Sequence type: amino acid Topology: linear Sequence type: protein Sequence Met Gly Leu Leu Arg Ile Met Met Pro Pro Lys Leu Gln Leu Leu Ala 1 5 10 15 Val Val Ala Phe Ala Val Ala Met Leu Phe Leu Glu Asn Gln Ile Gln 20 25 30 Lys Leu Glu Glu Ser Arg Ala Lys Leu Glu Arg Ala Ile Ala Arg His 35 40 45 Glu Val Arg Glu Ile Glu Gln Arg His Thr Met Asp Gly Pro Arg Gln 50 55 60 Asp Ala Ala Val Asp Glu Glu Glu Asp Ile Val Ile Ile Tyr Asn Arg 65 70 75 80 Val Pro Lys Thr Ala Ser Thr Ser Phe Thr Asn Ile Ala Tyr Asp Leu 85 90 95 Cys Ala Lys Asn Arg Tyr His Val Leu His Ile Asn Thr Thr Lys Asn 100 105 110 Asn Pro Val Met Ser Leu Gln Asp Gln Val Arg Phe Val Lys Asn Ile 115 120 125 Thr Thr Trp Asn Glu Met Lys Pro Gly Phe Tyr His Gly His Ile Ser 130 135 140 Tyr Leu Asp Phe Ala Lys Phe Gly Val Lys Lys Lys Pro Ile Tyr Ile 145 150 155 160 Asn Val Ile Arg Asp Pro Ile Glu Arg Leu Val Ser Tyr Tyr Tyr Tyr Phe 165 170 175 Leu Arg Phe Gly Asp Asp Tyr Arg Pro Gly Leu Arg Arg Arg Lys Gln 180 185 190 Gly Asp Lys Lys Thr Phe Asp Glu Cys Val Ala Glu Gly Gly Ser Asp 195 200 205 Cys Ala Pro Glu Lys Leu Trp Leu Gln Ile Pro Phe Phe Cys Gly His 210 215 220 Ser Ser Glu Cys Trp Asn Val Gly Ser Arg Trp Ala Met Asp Gln Ala 225 230 235 240 Lys Tyr Asn Leu Ile Asn Glu Tyr Phe Leu Val Gly Val Thr Glu Glu 245 250 255 Leu Glu Asp Phe Ile Met Leu Leu Glu Ala Ala Leu Pro Arg Phe Phe 260 265 270 Arg Gly Ala Thr Asp Leu Tyr Arg Thr Gly Lys Lys Ser His Leu Arg 275 280 285 Lys Thr Thr Glu Lys Lys Leu Pro Thr Lys Gln Thr Ile Ala Lys Leu 290 295 300 Gln Gln Ser Asp Ile Trp Lys Met Glu Asn Glu Phe Tyr Glu Phe Ala 305 310 315 320 Leu Glu Gln Phe Gln Phe Ile Arg Ala His Ala Val Arg Glu Lys Asp 325 330 335 Gly Asp Leu Tyr Ile Leu Ala Gln Asn Phe Phe Tyr Glu Lys Ile Tyr 340 345 350 Pro Lys Ser Asn 355

SEQ ID NO: 3 Sequence length: 2172 Sequence type: nucleic acid Number of strands: both forms Topology: linear Sequence type: cDNA Origin Organism name: human Tissue type: fetal brain Sequence characteristics Symbol indicating the: CDS Location: 355..1422 Method for determining characteristics: Features of P sequence Symbol indicating characteristics: transmembrane domain Location: 394..435 Method for determining features: Characteristics of P sequence Symbol indicating characteristics : Potential N-glycosylation site Location: 676..684 Method for determining characteristics: S sequence characteristics Symbol indicating characteristics: potential N-glycosylation site Location: 733..741 Characteristic determination method: S sequence GGGAAGGAAG GAAGAGAGGG AGGCGGGCAA GCAGGCGGGC GCGGGGGTCG GAGACTGAGG 60 CAGTAGAGGG AGGCGAGAGC CCGGCAGCCG CTTCGCGCTG TTTGCTGGCG CGGGTTTTGG 120 AGGGGGCGGC CGTTTAGTCG GCTGAGGAGA AGCGGACCT AGCGGCGTTG GTGATAGCGC 180 CTGGGGCT GG TGGCTGGCT GGG GGGACTGGCT GG GGCTCCTC CCGGCGTCTC TCTCGCCTCC 300 GGGGTCCCGC TCCCCGCCCC CCGCGGTATG TCTTGATCCC GAGCAGCGGG TTTC ATG 357 Met 1 GGG CTC CTC AGG ATT ATG ATG CCG CCC AAG TTG CAG CTG CTG GCG GTG 405 Gly Leu Leu Leu Aru Pro Glue Leu Arg Pro GTG GCC TTC GCG GTG GCG ATG CTC TTC TTG GAA AAC CAG ATC CAG AAA 453 Val Ala Phe Ala Val Ala Met Leu Phe Leu Glu Asn Gln Ile Gln Lys 20 25 30 CTG GAG GAG TCC CGC TCG AAG CTA GAA AGG GCT ATT GCA AGA CAC GAA 501 Leu Glu Glu Ser Arg Ser Lys Leu Glu Arg Ala Ile Ala Arg His Glu 35 40 45 GTC CGA GAA ATT GAG CAG CGA CAT ACA ATG GAT GGC CCT CGG CAA GAT 549 Val Arg Glu Ile Glu Gln Arg His Thr Met Asp Gly Pro Arg Gln Asp 50 55 60 65 GCC ACT TTA GAT GAG GAA GAG GAC ATG GTG ATC ATT TAT AAC AGA GTT 597 Ala Thr Leu Asp Glu Glu Glu Asp Met Val Ile Ile Tyr Asn Arg Val 70 75 80 CCC AAA ACG GCA AGC ACT TCA TTT ACC AAT ATC GCC TAT GAC CTG TGT 645 Pro Lys Thr Ala Ser Thr Ser Phe Thr Asn Ile Ala Tyr Asp Leu Cys 85 90 95 GCA AAG AAT AAA TAC CAT GTC CTT CAT ATC AAC ACT ACC AAA AAT AAT 693 Ala Lys Asn Lys Tyr His Val Leu His Ile Asn Thr Thr Lys Asn Asn 100 105 110 CCA GTG ATG TCA TTG CAA GAT CAG GTG CGC TTT GTA AAG AAT ATA ACT 741 Pro Val Met Ser Leu Gln Asp Gln Val Arg Phe Val Lys Asn Ile Thr 115 120 125 TCC TGG AAA GAG ATG AAA CCA GGA TTT TAT CAT GGA CAC GTT TCT TAC 789 Ser Trp Lys Glu Met Lys Pro Gly Phe Tyr His Gly His Val Ser Tyr 130 135 140 145 TTG GAT TTT GCA AAA TTT GGT GTG AAG AAG AAA CCA ATT TAC ATT AAT 837 Leu Asp Phe Ala Lys Phe Gly Val Lys Lys Lys Pro Ile Tyr Ile Asn 150 155 160 GTC ATA AGG GAT CCT ATT GAG AGG CTA GTT TCT TAT TAT TAC TTT CTG 885 Val Ile Arg Asp Pro Ile Glu Arg Leu Val Ser Tyr Tyr Tyr Phe Leu 165 170 175 AGA TTT GGA GAT GAT TAT AGA CCA GGG TTA CGG AGA CGA AAA CAA GGA 933 Arg Phe Gly Asp Asp Tyr Arg Pro Gly Leu Arg Arg Arg Lys Gln Gly 180 185 190 GAC AAA AAG ACC TTT GAT GAA TGT GTA GCA GAA GGT GGC TCA GAC TGT 981 Asp Lys Lys Thr Phe Asp Glu Cys Val Ala Glu Gly Gly Ser Asp Cys 195 200 205 GCT CCA GAG AAG CTC TGG CTT CAA ATC CCG TTC TTC TGT GGC CAT AGC 1029 Ala Pro Glu Lys Leu Trp Leu Gln Ile Pro Phe Phe Cys Gly His Ser 210 215 220 225 TCC GAA TGC TGG AAT GTG GGA AGC AGG TGG GCT ATG GAT CAA GCC AAG 1077 Ser Glu Cys Trp Asn Val Gly Ser Arg Trp Ala Met Asp Gln Ala Lys 230 235 240 TAT AAC CTA ATT AAT GAA TAT TTT CTG GTG GGA GTT ACT GAA GAA CTT 1125 Tyr Asn Leu Ile Asn Glu Tyr Phe Leu Val Gly Val Thr Glu Glu Leu 245 250 255 GAA GAT TTT ATC ATG TTA TTG GAG GCA GCA TTG CCC CGG TTT TTC AGG 1173 Glu Asp Phe Ile Met Leu Leu Glu Ala Ala Leu Pro Arg Phe Phe Arg 260 265 270 GGT GCT ACT GAA CTC TAT CGC ACA GGA AAG AAA TCTCAT CTT AGG AAA 1221 Gly Ala Thr Glu Leu Tyr Arg Thr Gly Lys Lys Ser His Leu Arg Lys 275 280 285 ACC ACA GAG AAG AAA CTC CCC ACT AAA CAA ACC ATT GCA AAA CTA CAG 1269 Thr Thr Glu Lys Lys Leu Pro Thr Lys Gln Thr Ile Ala Lys Leu Gln 290 295 300 305 CAA TCT GAT ATT TGG AAA ATG GAG AAT GAG TTC TAT GAA TTT GCA CTA 1317 Gln Ser Asp Ile Trp Lys Met Glu Asn Glu Phe Tyr Glu Phe Ala Leu 310 315 320 GAG CAG TTC CAA TTC ATC AGA GCC CAT GCC GTT CGA GAA AAA GAT GGA 1365 Glu Gln Phe Gln Phe Ile Arg Ala His Ala Val Arg Glu Lys Asp Gly 325 330 335 GAC CTC TAC ATC CTC GCA CAA AAC TTT TTC TAT GAA AAG ATT TAC CCT 1413 Asp Leu Tyr Ile Leu Ala Gln Asn Phe Phe Tyr Glu Lys Ile Tyr Pro 340 345 350 AAG TCG AAC TGAGTATAAG GTGTGACTAT TAGATTCTTG AACTAAAATT 1462 Lys Ser Asn 355 TGACCCTGTC TTCACCTTTG TTCTCAGCTC CACAGTCTGG ATTGCTGACA GTAGGTGTAT 1522 ATGACAATTT GTATTGAGCC AAATTAGGAA ACAGACAGTA ACGTCAAGGA AGTAGATACT 1582 GGCTGGCATT GTCAGTGTTC TAAGTTTCAG GCATTTTTAT TTTTTCCTGG CTAAACGTTG 1642 GTGAAAGTTA TAACCTCCTG CCTGGGAGAA AATATACATC ACCTAAAATG AACTTATGGC 1702 AGGTCTAATC AAAAGGCTAA ATACAATTTC AGAAAAGGTT CTGATACTCT TGTTTTTGAT 1762 AAAGCATTTT TTCAACTAAC CATGAATTAA GATGAGTCCA TTTGCCTCTT CTGCCTTCAC 1822 TGAGGGTTTG GGTTATACAC CTCTACTGAA TTGTGTTAAT AACTGTTTGG CAGTGTGTAC 1882 TTTGTTTTTG TGAGTCATGT CTCATGAAAT TTATTGGAAT GTTTAATCAT ATTTGCTAAG 1942 AAATGTTTCT GCTGTAGTTG GATTTGCCCA TATTTATGTA GGTGGTTTTA ATTTTTTAAA 2002 TG GTGATTAG TGTTAAAAAT CAATTTAAAT CATGACTAAT ATGGTAAAAA GATAAAGCAT 2062 CAAAGCAGTA TTTCTCATTC CTGCCTCCTC AATATCTAAT ACTGGGAAGA TACTTCAAAG 2122 AATATTGAGA TTGTCTGAAG TTTTAGTTAA GATTTTCACA CATTAATATC 2172

SEQ ID NO: 4 Sequence length: 356 Sequence type: amino acid Topology: linear Sequence type: protein sequence Met Gly Leu Leu Arg Ile Met Met Pro Lys Leu Gln Leu Leu Ala 1 5 10 15 Val Val Ala Phe Ala Val Ala Met Leu Phe Leu Glu Asn Gln Ile Gln 20 25 30 Lys Leu Glu Glu Ser Arg Ser Lys Leu Glu Arg Ala Ile Ala Arg His 35 40 45 Glu Val Arg Glu Ile Glu Gln Arg His Thr Met Asp Gly Pro Arg Gln 50 55 60 Asp Ala Thr Leu Asp Glu Glu Glu Asp Met Val Ile Ile Tyr Asn Arg 65 70 75 80 Val Pro Lys Thr Ala Ser Thr Ser Phe Thr Asn Ile Ala Tyr Asp Leu 85 90 95 Cys Ala Lys Asn Lys Tyr His Val Leu His Ile Asn Thr Thr Lys Asn 100 105 110 Asn Pro Val Met Ser Leu Gln Asp Gln Val Arg Phe Val Lys Asn Ile 115 120 125 Thr Ser Trp Lys Glu Met Lys Pro Gly Phe Tyr His Gly His Val Ser 130 135 140 Tyr Leu Asp Phe Ala Lys Phe Gly Val Lys Lys Lys Pro Ile Tyr Ile 145 150 155 160 Asn Val Ile Arg Asp Pro Ile Glu Arg Leu Val Ser Tyr Tyr Tyr Tyr Phe 165 170 175 Leu Arg Phe Gly Asp Asp Tyr Arg Pro Gly Leu Arg Arg Arg Lys Gln 180 185 190 Gly Asp Lys Lys Thr Phe Asp Glu Cys Val Ala Glu Gly Gly Ser Asp 195 200 205 Cys Ala Pro Glu Lys Leu Trp Leu Gln Ile Pro Phe Phe Cys Gly His 210 215 220 Ser Ser Glu Cys Trp Asn Val Gly Ser Arg Trp Ala Met Asp Gln Ala 225 230 235 240 Lys Tyr Asn Leu Ile Asn Glu Tyr Phe Leu Val Gly Val Thr Glu Glu 245 250 255 Leu Glu Asp Phe Ile Met Leu Leu Glu Ala Ala Leu Pro Arg Phe Phe 260 265 270 Arg Gly Ala Thr Glu Leu Tyr Arg Thr Gly Lys Lys Ser His Leu Arg 275 280 285 Lys Thr Thr Glu Lys Lys Leu Pro Thr Lys Gln Thr Ile Ala Lys Leu 290 295 300 Gln Gln Ser Asp Ile Trp Lys Met Glu Asn Glu Phe Tyr Glu Phe Ala 305 310 315 320 Leu Glu Gln Phe Gln Phe Ile Arg Ala His Ala Val Arg Glu Lys Asp 325 330 335 Gly Asp Leu Tyr Ile Leu Ala Gln Asn Phe Phe Tyr Glu Lys Ile Tyr 340 345 350 Pro Lys Ser Asn 355

SEQ ID NO: 5 Sequence length: 13 Sequence type: Amino acid Topology: Single chain Sequence type: Peptide sequence Asp Leu Cys Ala Lys Asn Arg Tyr His Val Leu His Ile 1 5 10

SEQ ID NO: 6 Sequence length: 9 Sequence type: amino acid Topology: single chain Sequence type: peptide sequence Asp Gln Val Arg Phe Val Lys Asn Ile 15

SEQ ID NO: 7 Sequence length: 9 Sequence type: amino acid Topology: single chain Sequence type: peptide sequence Asp Xaa Tyr Arg Pro Gly Leu Xaa Arg 15

SEQ ID NO: 8 Sequence length: 8 Sequence type: amino acid Topology: single chain Sequence type: peptide sequence Asp Ile Val Ile Xaa Tyr Asn Arg 15

SEQ ID NO: 9 Sequence length: 4 Sequence type: amino acid Topology: single chain Sequence type: peptide Sequence Asp Leu Tyr Arg 1

SEQ ID NO: 10 Sequence length: 20 Sequence type: nucleic acid Topology: number of linear chains: single-stranded Sequence type: other nucleic acid Synthetic DNA sequence GCNAARAAYM GNTAYCAYGT 20

SEQ ID NO: 11 Sequence length: 20 Sequence type: Nucleic acid Topology: Number of linear chains: Single-stranded Sequence type: Other nucleic acids Synthetic DNA sequence MGNTAYCAYG TNYTNCAYAT 20

SEQ ID NO: 12 Sequence length: 20 Sequence type: Nucleic acid Topology: Number of linear chains: Single-stranded Sequence type: Other nucleic acids Synthetic DNA sequence TTYTTNACRA ANCKNACYTG 20

SEQ ID NO: 13 Sequence length: 20 Sequence type: Nucleic acid Topology: Number of linear chains: Single-stranded Sequence type: Other nucleic acids Synthetic DNA sequence TTNACRAANC KNACYTGRTC 20

SEQ ID NO: 14 Sequence length: 356 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Met Gly Leu Leu Arg Ile Met Met Pro Lys Leu Gln Leu Leu Ala 1 5 10 15 Val Val Ala Phe Ala Val Ala Met Leu Phe Leu Glu Asn Gln Ile Gln 20 25 30 Lys Leu Glu Glu Ser Arg Xaa Lys Leu Glu Arg Ala Ile Ala Arg His 35 40 45 Glu Val Arg Glu Ile Glu Gln Arg His Thr Met Asp Gly Pro Arg Gln 50 55 60 Asp Ala Xaa Xaa Asp Glu Glu Glu Asp Xaa Val Ile Ile Tyr Asn Arg 65 70 75 80 Val Pro Lys Thr Ala Ser Thr Ser Phe Thr Asn Ile Ala Tyr Asp Leu 85 90 95 Cys Ala Lys Xaa Arg Tyr His Val Leu His Ile Asn Thr Thrs Asn 100 105 110 Asn Pro Val Met Ser Leu Gln Asp Gln Val Arg Phe Val Lys Asn Ile 115 120 125 Thr Xaa Trp Xaa Glu Met Lys Pro Gly Phe Tyr His Gly His Xaa Ser 130 135 140 Tyr Leu Asp Phe Ala Lys Phe Gly Val Lys Lys Lys Pro Ile Tyr Ile 145 150 155 160 Asn Val Ile Arg Asp Pro Ile Glu Arg Leu Val Ser Tyr Tyr Tyr Tyr Phe 165 170 175 Leu Arg Phe Gly As p Asp Tyr Arg Pro Gly Leu Arg Arg Arg Lys Gln 180 185 190 Gly Asp Lys Lys Thr Phe Asp Glu Cys Val Ala Glu Gly Gly Ser Asp 195 200 205 Cys Ala Pro Glu Lys Leu Trp Leu Gln Ile Pro Phe Phe Cys Gly His 210 215 220 Ser Ser Glu Cys Trp Asn Val Gly Ser Arg Trp Ala Met Asp Gln Ala 225 230 235 240 Lys Tyr Asn Leu Ile Asn Glu Tyr Phe Leu Val Gly Val Thr Glu Glu 245 250 255 Leu Glu Asp Phe Ile Met Leu Leu Glu Ala Ala Leu Pro Arg Phe Phe 260 265 270 270 Arg Gly Ala Thr Xaa Leu Tyr Arg Thr Gly Lys Lys Ser His Leu Arg 275 280 285 Lys Thr Thr Glu Lys Lys Leu Pro Thr Lys Gln Thr Ile Ala Lys Leu 290 295 300 Gln Gln Ser Asp Ile Trp Lys Met Glu Asn Glu Phe Tyr Glu Phe Ala 305 310 315 320 Leu Glu Gln Phe Gln Phe Ile Arg Ala His Ala Val Arg Glu Lys Asp 325 330 335 Gly Asp Leu Tyr Ile Leu Ala Gln Asn Phe Phe Tyr Glu Lys Ile Tyr 340 345 350 Pro Lys Ser Asn 355

[Brief description of the drawings]

FIG. 1 shows a partial amino acid sequence of HS2ST and a base sequence of a primer for PCR.

FIG. 2 is a hydropathic plot of the amino acid sequence of Chinese hamster HS2ST predicted from the cDNA sequence.

FIG. 3 is a comparison of HS2ST cDNA derived from Chinese hamster and cDNA derived from human HS2ST.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12N 9/10 C12R 1:91)

Claims (14)

[Claims] 1. It has the amino acid sequence shown in SEQ ID NO: 14, and converts a sulfate group from a sulfate group donor into an L-iduronic acid residue contained in glycosaminoglycan which is a sulfate group acceptor.
At least one of the polypeptides of glycosaminoglycan sulfate transferase which may have substitution, deletion, insertion or rearrangement of one or more amino acid residues which do not substantially impair the enzymatic activity of transferring to the hydroxyl group. DNA having a base sequence that partially codes. 2. The DNA according to claim 1, which has a base sequence encoding at least a part of the amino acid sequence shown in SEQ ID NO: 14. 3. It has the amino acid sequence shown in SEQ ID NO: 2,
One or more amino acid residues that do not substantially impair the enzymatic activity of transferring a sulfate group from a sulfate group donor to a hydroxyl group at position 2 of an L-iduronic acid residue contained in glycosaminoglycan that is a sulfate group acceptor. A DNA having a nucleotide sequence encoding at least a part of a glycosaminoglycan sulfotransferase polypeptide which may have a substitution, deletion, insertion or transposition of a group. 4. The DNA according to claim 3, which has a base sequence encoding at least a part of the amino acid sequence shown in SEQ ID NO: 2. 5. It has the amino acid sequence shown in SEQ ID NO: 4,
One or more amino acid residues that do not substantially impair the enzymatic activity of transferring a sulfate group from a sulfate group donor to a hydroxyl group at position 2 of an L-iduronic acid residue contained in glycosaminoglycan that is a sulfate group acceptor. A DNA having a nucleotide sequence encoding at least a part of a glycosaminoglycan sulfotransferase polypeptide which may have a substitution, deletion, insertion or transposition of a group. 6. The amino acid sequence represented by SEQ ID NO: 4 having a base sequence encoding at least a part of the amino acid sequence represented by amino acids 1 to 356.
The described DNA. 7. A DNA having a nucleotide sequence encoding a glycosaminoglycan sulfotransferase polypeptide having the following properties: Action: A sulfate group is selectively transferred from a sulfate group donor to a hydroxyl group at the 2-position of an L-iduronic acid residue contained in glycosaminoglycan which is a sulfate group acceptor. Substrate specificity: transfers sulfate groups to heparan sulfate and CDSNS-heparin, but does not transfer sulfate groups to chondroitin, chondroitin sulfate, dermatan sulfate and keratan sulfate. Optimum reaction pH: around pH 5.0 to 6.5 Optimum reaction ionic strength: around 50 to 200 mM (in the case of sodium chloride) Inhibition and activation Enzyme activity is increased by coexisting protamine. Adenosine-3 ', 5'-diphosphate (3', 5'-A
Enzyme activity is inhibited by the coexistence of DP).
Coexistence of 10 mM or less of dithiothreitol (DTT) hardly affects the enzyme activity. Molecular weight: molecular weight estimated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions after N-glycosidase treatment: about 38,000. 8. The DNA according to claim 7, which is derived from Chinese hamster or human. 9. It has the amino acid sequence shown in SEQ ID NO: 14, and converts a sulfate group from a sulfate group donor into an L-iduronic acid residue contained in glycosaminoglycan that is a sulfate group acceptor.
All of the glycosaminoglycan sulfate transferase polypeptides which may have substitution, deletion, insertion or rearrangement of one or more amino acid residues which do not substantially impair the enzymatic activity of transferring to the hydroxyl group at position 1. Or a polypeptide consisting of a moiety. 10. It has an amino acid sequence represented by SEQ ID NO: 2, and converts a sulfate group from a sulfate group donor into an L-iduronic acid residue 2 contained in glycosaminoglycan that is a sulfate group acceptor.
All of the glycosaminoglycan sulfate transferase polypeptides which may have substitution, deletion, insertion or rearrangement of one or more amino acid residues which do not substantially impair the enzymatic activity of transferring to the hydroxyl group at position 1. Or a polypeptide consisting of a moiety. 11. It has an amino acid sequence represented by SEQ ID NO: 4, and converts a sulfate group from a sulfate group donor to a 2-sulfonate residue of L-iduronic acid contained in glycosaminoglycan which is a sulfate group acceptor.
All of the glycosaminoglycan sulfate transferase polypeptides which may have substitution, deletion, insertion or rearrangement of one or more amino acid residues which do not substantially impair the enzymatic activity of transferring to the hydroxyl group at position 1. Or a polypeptide consisting of a moiety. 12. It has an amino acid sequence represented by SEQ ID NO: 4, and converts a sulfate group from a sulfate group donor into an L-iduronic acid residue contained in glycosaminoglycan which is a sulfate group acceptor.
A glycosaminoglycan sulfotransferase comprising a polypeptide which may have one or more amino acid residue substitutions, deletions, insertions or rearrangements which do not substantially impair the enzymatic activity of transferring to a hydroxyl group at the position. 13. A polypeptide comprising all or a part of the glycosaminoglycan sulfate transferase polypeptide encoded by the nucleotide sequence of the DNA according to claim 7 or 8. 14. A cell transformed with the DNA according to any one of claims 1 to 8, which is cultured in a suitable medium,
A method for producing a polypeptide, comprising producing and accumulating a glycosaminoglycan sulfotransferase polypeptide encoded by a base sequence of the DNA in a culture, and collecting the polypeptide from the culture.