JP2003299488A - Fusion protein having glucuronic acid transferase activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fusion protein useful in obtaining in a genetically engineering way an enzyme substantially having no N-galactosamine transferase activity but having glucuronic acid transferase activity among enzymes involved in the synthesis of chondroitin skeleton, to provide a vector for obtaining such a fused protein, and to provide a transformant obtained by introducing the vector into a host cell. <P>SOLUTION: The fusion protein is such one that a discriminant peptide and a polypeptide are fused together, wherein the polypeptide is constituted of an amino acid sequence described in the sequence No.4 (See the Specification) or an amino acid sequence where at least one constituent amino acid is substituted, deleted, inserted or translocated in the said amino acid sequence. This fusion protein has the activity of transferring glucuronic acid onto the N-acetylgalactosamine residue on a nonreduced terminal present in a chondroitin fundamental skeleton. The vector containing a nucleic acid encoding this fusion protein, and the transformant are also provided respectively. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a fusion protein having an enzymatic activity, more specifically, a fusion protein having an enzymatic activity of transferring glucuronic acid to an N-acetylgalactosamine residue at the non-reducing end present in the chondroitin skeleton. Regarding proteins.

[0002]

BACKGROUND OF THE INVENTION Chondroitin sulfate and chondroitin are D-glucuronic acids (hereinafter simply referred to as "glucuronic acid" or "Gl
cA) residue and N-acetylgalactosamine (hereinafter simply “N-acetylgalactosamine” or “GalNA”).
(also referred to as “c”) residue disaccharide repeating structure (ie,-[GlcAβ (1,3) -GalNAcβ (1,4)-] _n : n is an integer of 2 or more) It is a type of glycosaminoglycan.

In recent years, glycosaminoglycans, particularly chondroitin and chondroitin sulfate, have been conventionally extracted and purified from animal organs and the like, but due to lack of raw materials, due to the repeating structure of the above disaccharide common to chondroitin and chondroitin sulfate. A method for artificially synthesizing a basic skeleton (also called a chondroitin skeleton) is being sought. In particular, if it is a human-derived enzyme, even if artificially synthesized chondroitin or chondroitin sulfate is contaminated with the enzyme, a biological defense mechanism such as an antigen-antibody reaction does not act strongly. An enzyme for synthesizing such a basic skeleton, among them, an enzyme derived from human, having glucuronosyltransferase activity, and not having N-acetylgalactosaminyltransferase activity has not been found yet.

[Non-patent document] J. Biol. Chem., 277 (2002), pp.38179.
-38188

[0004]

Among human-derived enzymes involved in the synthesis of chondroitin skeleton, a large amount of enzymes having glucuronosyltransferase activity but substantially no N-acetylgalactosaminyltransferase activity. Although a gene encoding the enzyme was needed for synthesis, and such a gene was also needed to explore the possibility of gene therapy, the gene for such enzyme was required. Was not obtained.

Therefore, an object of the present invention is, among enzymes involved in the synthesis of chondroitin skeleton, an enzyme having substantially no N-acetylgalactosaminyltransferase activity but having glucuronosyltransferase activity (hereinafter also referred to as "GlcAT"). (Abbreviated)
It is intended to provide a fusion protein of a peptide and a discriminating peptide, a method for producing the same by genetic engineering and a method for producing a sugar chain having a chondroitin skeleton.

[0006]

Means for Solving the Problems As a result of extensive studies to achieve the above object, the present inventor found a cDNA for human GlcAT and completed the present invention by enabling large-scale synthesis of human GlcAT.

That is, the present invention is as follows. (1) The amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 4
A polypeptide consisting of an amino acid sequence having one or more constituent amino acid substitutions, deletions, insertions, or transpositions in the described amino acid sequence, and a discriminant peptide fused to each other and present in the chondroitin skeleton from the glucuronic acid donor. A fusion protein having an enzyme activity of transferring glucuronic acid to an N-acetylgalactosamine residue at a non-reducing end. (2) The non-reducing terminal is an N-acetylgalactosamine residue from the glucuronic acid donor, and the glucuronic acid residue is transferred to the N-acetylgalactosamine of the oligosaccharide consisting of 7 sugars having a chondroitin skeleton, Activity of transferring an N-acetylgalactosamine residue from an N-acetylgalactosamine donor to a glucuronic acid of an oligosaccharide consisting of a hexasaccharide having a chondroitin skeleton and having a non-reducing end as a glucuronic acid residue The fusion protein according to (1), which does not have: (3) Discrimination peptide is signal peptide, protein kinase A, protein A, glutathione S transferase, His
Tag, myc tag, FLAG protein, T7 tag, S tag, HSV
Tags, pelB, HA tags, Trx tags, CBP tags, CBD tags, CBR
Tags, β-lac / blu, β-gal, luc, HP-Thio, HSP, Ln
γ, Fn, GFP, YFP, CFP, BFP, DsRed, DsRed2, MBP, La
The fusion protein according to (1) or (2), which is any one peptide selected from the group consisting of cZ, IgG, avidin, and protein G. (4) A DNA encoding the fusion protein according to any one of (1) to (3). (5) A vector containing the DNA according to (4). (6) A transformant obtained by introducing the vector according to (5) into a host cell. (7) The transformant according to (6) is grown, and the fusion protein is isolated from the grown product (1) to
(3) The method for producing the fusion protein according to any one of the above. (8) An antibody which specifically recognizes the fusion protein according to any one of (1) to (3). (9) A glucuronosyltransferase having the following properties. (A) Action The glucuronic acid residue is transferred from the glucuronic acid donor to the acceptor N-acetylgalactosamine residue. (B) From the substrate-specific glucuronic acid donor, the non-reducing end is an N-acetylgalactosamine residue and has a chondroitin skeleton 7
It transfers a glucuronic acid residue to the N-acetylgalactosamine of the oligosaccharide consisting of sugar, but from the N-acetylgalactosamine donor, the non-reducing end is a glucuronic acid residue and consists of a hexasaccharide having a chondroitin skeleton. The oligosaccharide has substantially no activity to transfer the N-acetylgalactosamine residue to the glucuronic acid. (C) Inhibition of activity In the coexistence of ethylenediaminetetraacetic acid, it exhibits substantially no enzyme activity. (10) Substitution of one or more constituent amino acids in the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4,
The glucuronosyltransferase according to (9), which comprises an amino acid sequence having a deletion, an insertion, or a transposition. (11) The fusion protein according to any one of (1) to (3) or the glucuronic acid according to (9) or (10) for a sugar chain having an N-acetylgalactosamine residue at a non-reducing end and a chondroitin skeleton. The glucuronic acid donor is allowed to act on the transferase and the non-reducing end of the sugar chain
A method for producing a sugar chain having a chondroitin skeleton, which comprises transferring glucuronic acid to an N-acetylgalactosamine residue. (12) Substitution of one or more constituent amino acids in the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4,
It contains a protein containing a polypeptide consisting of an amino acid sequence having a deletion, insertion, or rearrangement, and has an N-terminal at the non-reducing end.
A sugar chain synthesizing agent having an activity of transferring glucuronic acid from a glucuronic acid donor to a sugar chain having a chondroitin skeleton having an acetylgalactosamine residue. (13) A fusion of a polypeptide consisting of an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence of SEQ ID NO: 4 having one or more constituent amino acid substitutions, deletions, insertions, or transpositions with a discriminating peptide The sugar chain synthesizing agent according to (12), which contains a protein.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below with reference to embodiments of the invention. (1) Enzyme of the present invention The enzyme of the present invention has the following properties. (A) Action The glucuronic acid residue is transferred from the glucuronic acid donor to the acceptor N-acetylgalactosamine residue. (B) From the substrate-specific glucuronic acid donor, the non-reducing end is an N-acetylgalactosamine residue and has a chondroitin skeleton 7
It transfers a glucuronic acid residue to the N-acetylgalactosamine of the oligosaccharide consisting of sugar, but from the N-acetylgalactosamine donor, the non-reducing end is a glucuronic acid residue and consists of a hexasaccharide having a chondroitin skeleton. The oligosaccharide has substantially no activity to transfer the N-acetylgalactosamine residue to the glucuronic acid. (C) Inhibition of activity In the absence of a divalent metal cation, it exhibits substantially no enzyme activity.

Examples of the glucuronic acid donor in the enzyme of the present invention include glucuronic acid nucleotides.
ADP (adenosine diphosphate) -GlcA, UDP (uridine diphosphate) -GlcA, CDP (cytidine diphosphate) -GlcA, GDP (guanosine diphosphate) -GlcA, etc. are exemplified. UDP- which generally acts as a glucuronic acid donor in
GlcA is most preferred.

An oligosaccharide consisting of 7 sugars having a non-reducing end as an N-acetylgalactosamine residue and having a chondroitin skeleton means, for example, chondroitin derived from whale cartilage or shark cartilage by testicular hyaluronidase as described in Examples. Using chondroitin sulfate hexasaccharide purified by limited decomposition of sulfuric acid as a substrate, in the presence of an N-acetylgalactosamine donor, a chondroitin synthase derived from Escherichia coli K4 was allowed to act to transfer N-acetylgalactosamine to a non-reducing end. It can be prepared.

The N-acetylgalactosamine donor is N-
Acetylgalactosamine nucleotides such as ADP-GalNA
c, UDP-GalNAc, CDP-GalNAc, GDP-GalNAc and the like are exemplified, and among them, UDP-GalNAc which generally acts as an N-acetylgalactosamine donor in vivo is most preferably exemplified. The non-reducing end is a glucuronic acid residue, and an oligosaccharide consisting of 6 sugars having a chondroitin skeleton, and as described in Examples, whale cartilage-derived or shark cartilage-derived chondroitin sulfate is limitedly decomposed by testicular hyaluronidase. Chondroitin sulfate 6 obtained by purification
A sugar is illustrated.

The activity of transferring a glucuronic acid residue and the activity of transferring an N-acetylgalactosamine residue are determined by, for example, the activity of each donor (preferably carbon isotope: ¹⁴ C, ³ H (tritium), etc.) Labeled with a fluorescent substance (radioactivity is preferable because it does not cause steric hindrance to the substrate), the enzyme is reacted using this donor, and the reaction product is labeled with a liquid scintillation counter, autoradiography, or the like. It is possible to detect the enzyme activity by analyzing with a method for detecting a substance. These activities can be measured according to the method described in <4> in Example 1. The term "substantially free of activity" is used to mean that even when the measurement is performed according to the method described in <4> of Example 1, only the same level of activity as that of the negative control is observed.

The effect of the divalent metal cation on the enzymatic reaction is, for example, as described in Examples, the enzymatic reaction when a divalent metal cation, preferably manganese ion (Mn ²⁺ ) coexists in the reaction system. It can be easily compared by comparing the reaction system with an enzyme reaction in a reaction system in which a chelating agent for a metal ion such as ethylenediaminetetraacetic acid (hereinafter also referred to as “EDTA”) is added to the reaction system. .

The enzyme of the present invention comprises the amino acid sequence of SEQ ID NO: 4, since it is clear from the examples below that the amino acid sequence of SEQ ID NO: 4 has the same enzymatic activity when it is used as a fusion protein. Preferably, the amino acid sequence of SEQ ID NO: 4 may further have a transmembrane region composed of 10 to 20 hydrophobic amino acid residues. However, when the enzyme of the present invention is prepared by a genetic engineering technique, the transmembrane region consisting of a hydrophobic amino acid region requires complicated work for purification / isolation of the enzyme of the present invention. , A polypeptide consisting only of the amino acid sequence set forth in SEQ ID NO: 4 or a sugar polypeptide having a sugar chain bonded thereto.

(2) Substance of the present invention The substance of the present invention is a fusion of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 and the discriminating peptide, and the residue of N-acetylgalactosamine at the non-reducing end present in the chondroitin skeleton It is a fusion protein having an activity of transferring a glucuronic acid residue to a base.

Here, the discriminating peptide is a peptide used for facilitating the secretion, separation, purification and detection of the fusion protein from the growth product of the transformant when the fusion protein is prepared by gene recombination. , Eg signal peptides (present at the N-terminus of many proteins and functioning intracellularly for protein selection)
Peptides consisting of 15 to 30 amino acid residues: eg OmpA, Om
pT, Dsb, etc.), protein kinase A, protein A (a component of Staphylococcus aureus cell wall and having a molecular weight of about 42,000), glutathione S-transferase, His tag (6 to 10 histidine residues arranged side by side), myc tag (13 amino acid sequence derived from cMyc protein), FLAG peptide (analytical marker consisting of 8 amino acid sequence), T7 tag (gene10
First 11 amino acid sequence of protein), S tag (pancreatic RNa
seA-derived 15 amino acid sequence), HSV tag, pelB (22 amino acid sequence of Escherichia coli outer membrane protein pelB), HA tag (10-amino acid sequence derived from hemagglutinin), Trx tag (thioredoxin sequence), CBP tag (calmodulin-binding peptide), CBD tag (cellulose binding domain), CBR tag (collagen binding domain), β-lac / blu (β lactamase), β-gal (β galactosidase), luc (luciferase), HP-Thio (His-patch thioredoxin), HS
P (heat shock peptide), Lnγ (laminin γ peptide), Fn (fibronectin partial peptide), GFP (green fluorescent peptide), YFP (yellow fluorescent peptide), CFP (cyan fluorescent peptide), BFP (blue fluorescent peptide), DsRe
d, DsRed2 (red fluorescent peptide), MBP (maltose binding peptide), LacZ (lactose operator), IgG
Any peptide selected from the group consisting of (immunoglobulin G), avidin, and protein G is referred to, and any discriminating peptide can be used. Among them, especially signal peptides and protein kinases
A, protein A, glutathione S transferase, His tag, my
c tag, FLAG peptide, T7 tag, S tag, HSV tag, pelB
Alternatively, the HA tag is preferable because it facilitates expression and purification of the substance of the present invention by a genetic engineering method.

The arrangement of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 and the discriminating peptide in the substance of the present invention is such that the discriminating peptide is bound to the C-terminal side of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 4. Alternatively, it may be bound to the N-terminal side. Even if such a bond is made through a spacer composed of an amino acid sequence having no physiological activity (a peptide consisting of about 2 to 10 amino acid sequences), the substance of the present invention is present in the basic skeleton of chondroitin. There is no limitation as long as it has an activity of transferring a glucuronic acid residue to an N-acetylgalactosamine residue (hereinafter, also referred to as “GlcAT activity”).

Here, the GlcAT activity is, for example, Kitagawa et al.
It is possible to detect by using a method (described in Examples below) in which al., J. Biol. Chem., 276 (2001), 38721-38726 is modified. When the GlcAT activity of the substance of the present invention is measured by such a method, the Michaelis constant (K
m value) is preferably 100 μmol / l or less, particularly preferably 90 μmol / l or less, particularly 80 μmol / l with respect to “11-sugar derived from chondroitin sulfate (CS11)” described in Examples below. It is preferably 1 or less. Also,
Furthermore, UDP-GlcUA, which is a preferred example of a glucuronic acid donor,
The Km value for is preferably 100 μmol / l or less, more preferably 95 μmol / l, most preferably 90 μmol / l or less. Such extremely highly specific GlcAT activity is considered to be one of the surprising effects due to the fact that the substance of the present invention has a form in which the specific amino acid sequence of SEQ ID NO: 4 is a fusion protein.

The binding mode of glucuronic acid in the “transfer activity of glucuronic acid residue to N-acetylgalactosamine residue” possessed by the substance of the present invention is as follows: the 3-position hydroxyl group of N-acetylgalactosamine residue of the chondroitin skeleton and glucuronic acid. Since it is a β1-3 glycoside bond with the 1-position hydroxyl group of the residue, it is not particularly limited, but is preferably a β1-3 glycoside bond.

In general, the enzymatic activity can be maintained even if one or more (usually 2 or more and 50 or less) constituent amino acids in the amino acid sequence of the enzyme protein are substituted, deleted, inserted, and / or transposed. It is known and can be said to be a variant of the same enzyme, but also in the substance of the present invention, one or more (2 or more and 50 or less) in the amino acid sequence of SEQ ID NO:
Even if a partial mutation such as substitution, deletion, insertion, and / or rearrangement of the constituent amino acids of GlcA
It can be said that the substance is substantially the same as the substance of the present invention as long as it retains T activity (such a polypeptide having a partial mutation in the polypeptide consisting of the amino acid sequence of SEQ ID NO: 4). Is referred to as a "modified polypeptide" for convenience). It is preferable that the amino acid sequence of such a modified polypeptide has a homology of 93% or more, preferably 95% or more, further 97% or more with the amino acid sequence shown in SEQ ID NO: 4 (that is, the number of amino acid mutations is A maximum of 50, 36 or less is preferable, and 22 or less is most preferable. The amino acid sequence homology is FA
It can be easily calculated using a well-known computer software such as STA, and such software is also available on the Internet.

The above-mentioned fusion protein has the amino acid sequence as described above, and a sugar chain may be bound to the protein as long as it has the above-mentioned enzymatic activity. That is, the fusion protein of the substance of the present invention includes a fusion protein in the form of glycoprotein.

Such a substance of the present invention uses a glycosaminoglycan having a chondroitin skeleton as a substrate and transfers a glucuronic acid residue to a galactosamine residue at the non-reducing end in the presence of a glucuronic acid donor, which will be described later. It can be used as an active ingredient of the "glycan synthesizer of the present invention".

Further, since the enzyme activity of the substance of the present invention can be easily measured according to the method described in Examples below, for example, a test substance is added to the assay system for the enzyme activity, and A method of measuring / determining whether or not to control (enhance or suppress) the enzyme activity of the invented substance, and selecting a test substance having a function of controlling the enzyme activity, that is, "a substance that controls glucuronosyltransferase activity" It is possible to use the substance of the present invention in a "screening method".

(3) Method for Preparing Substance of the Present Invention The substance of the present invention has a substitution, deletion, insertion, or transposition of one or more constituent amino acids in the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4. The DNA encoding the amino acid sequence and the DNA encoding the discriminating peptide are arranged in the same reading region, and the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 has one or more constituent amino acids substituted or deleted. Loss, insertion, or by transposing a suitable host cell transfected with a vector having a promoter region capable of expressing a polypeptide consisting of an amino acid sequence having transposition and a discrimination peptide,
It is possible to carry out the preparation by expressing the GlcAT gene and allowing it to accumulate in the growth product. The host cell here is a prokaryotic cell (for example, E. coli, if it can express the above gene).
Bacillus subtilis and the like) are not limited to eukaryotic cells (eg yeast, cells of mammalian origin, cells of insect origin, etc.). In particular, since the polypeptide having the amino acid sequence of SEQ ID NO: 4 is a polypeptide originally expressed in eukaryotic cells, eukaryotic cells are preferred as host cells, and mammalian cells are particularly preferred.

Here, the growth of the host cell includes culturing the host cell as well as administering the host cell to a living body or the like and growing the host cell in the living body. In addition to the cultured host cells and culture supernatant, the growth product also includes excrements and secretions of the living body when the host cells are grown in vivo.

The present inventors have conducted Ge
Since it has been clarified that the cDNA of nBank Accession No. AB037823 (consisting of the nucleotide sequence of SEQ ID NO: 1) is a DNA encoding human GlcAT, the substance of the present invention can be prepared based on the cDNA. . The above cDNA is pBluescr
It is stored in the Kazusa DNA Research Institute (Kazusa Kamafoot, Kisarazu City, Chiba Prefecture) in the form of being inserted into the iptII SK (+) plasmid and can be sold.

As the identification peptide of the substance of the present invention, for example, FL
When AG peptides are used and mammalian cells are used as host cells, for example, pFLAG-CMV-3 vector (manufactured by Sigma) can be used as an expression vector.

In order to insert the above cDNA inserted into pBluescriptII SK (+) plasmid into pFLAG-CMV-3 vector, appropriate restriction enzymes such as NarI and BamHI are used from pBluescriptII SK (+) plasmid vector. CDN
A is cut out and the sticky ends of the cDNA are blunted to obtain a cDNA fragment (consisting of the nucleotide sequence of SEQ ID NO: 3), pFLAG-CM
PF treated with an appropriate restriction enzyme by ligating an appropriate restriction end such as HindIII for insertion into a V-3 vector
It can be inserted into the LAG-CMV-3 vector.

The pFLAG-CMV-3 vector (pFLAGGlcAT) thus prepared is used in COS-1 cells or COS-7 cells functioning as host cells for the pFLAG-CMV-3 vector by electroporation or the like. A transformant can be obtained by using the method.

The transformant can be grown under conditions suitable for growing the host cell on which it is based, and the substance of the present invention can be prepared from the grown product. For example, when COS-7 cells are selected as host cells, the transformant is cultured in vitro, and the substance of the present invention is isolated and purified from the culture (the transformant and the culture supernatant after the culture). It is possible. When the pFLAG-CMV-3 vector is used, the substance of the present invention will contain a FLAG peptide, so the substance of the present invention uses, for example, an anti-FLAG antibody or the antibody of the present invention described below,
For example, the substance of the present invention can be isolated and purified by a method such as affinity purification. The method of isolation and purification is
Depending on the discriminating peptide in the substance of the present invention, an appropriate method can be routinely selected.

The discriminating peptide has the amino acid sequence specifically recognized by collagenase or the like (for example, the amino acid sequence described in SEQ ID NO: 3 of JP-A No. 11-137256, etc.), and thus the sequence described in SEQ ID NO: 4 is obtained. 1 in the amino acid sequence or the amino acid sequence of SEQ ID NO: 4
It is possible to obtain the enzyme of the present invention described in (1), which contains an amino acid sequence having the substitution, deletion, insertion, or transposition of the above constituent amino acids.

(4) Antibody of the Present Invention The antibody of the present invention is an antibody characterized by specifically binding to the substance of the present invention. The antibody of the present invention specifically binds to the substance of the present invention via the antigen-binding site of the antibody. Therefore, in producing the antibody of the present invention, it is possible to use a known method using the substance of the present invention as an "immunogen". More specifically, the substance of the present invention is used to immunize animals such as rabbits, mice, rats, goats, sheep, dogs, cats, horses and monkeys, and sera of these animals are collected to obtain antiserum (polyclonal Antibodies), antibody-producing cells (spleen cells, etc.) are collected from these animals and fused with cells having a continuous proliferation ability such as myeloma cell lines (myeloma cells) to obtain fused cells ( A monoclonal antibody can be efficiently produced by obtaining a hybridoma) and culturing them or proliferating them in the abdominal cavity of an animal. Such hybridoma can be easily prepared by those skilled in the art based on the present specification.

The antibody of the present invention has the ability to specifically recognize and bind to the substance of the present invention, using the substance of the present invention or a part thereof as an antigenic determinant or an epitope (these are collectively referred to as “epitope etc.”). Have. Therefore, the antibody of the present invention can be used in an in vitro or in vivo assay in which a fragment of the substance of the present invention containing the above-mentioned epitope or the like or a substance of the present invention is detected. Further, the antibody of the present invention can also be used when purifying a fragment of the substance of the present invention containing an epitope or the like or a substance of the present invention by immunoaffinity chromatography.

The above-mentioned epitope or the like may be linear or conformational (intermittent).
The epitope and the like can be identified by a conventional method known in the art. Therefore, one of the
One aspect relates to an antigenic epitope of a fusion protein of the glucuronosyltransferase protein of the present invention and a discriminating peptide. Such epitopes are useful in raising antibodies, particularly monoclonal antibodies, as described in more detail below. Furthermore, since the part of the epitope contained in the substance of the present invention specifically binds to the antibody of the present invention, it can be used in an assay for measuring the abundance of the antibody of the present invention in a living body or in a sample.

The antibody of the present invention can be prepared using the substance of the present invention or a part thereof, or the above-mentioned epitope portion. The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody, and both can be prepared by a conventional technique. For example, Kennet et al. (Supervised), Monoclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Plenum Press, N
With reference to ew York, 1980, those skilled in the art can easily prepare the antibody of the present invention. However, it is preferably a monoclonal antibody because of its versatility.

The antibody of the present invention which is a monoclonal antibody is
Chimeric antibodies (such as humanized antibodies of murine monoclonal antibodies and humanized antibodies of monkey monoclonal antibodies are exemplified) are included. The preparation of such humanized antibodies by known techniques has the advantage of being less immunogenic when administered to humans. Also included in the invention are antigen binding fragments of antibodies that can be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab and F (ab ′) ₂ fragments, for example. In addition, antibody fragments and derivatives produced by genetic engineering techniques are also exemplified.

(5) Sugar chain synthesizing agent of the present invention The sugar chain synthesizing agent of the present invention contains a protein containing a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4, and has an N-acetylgalactosamine residue at the non-reducing end. It has an activity of transferring glucuronic acid from a glucuronic acid donor to a sugar chain composed of a chondroitin skeleton.

The protein contained in the sugar chain synthesizing agent of the present invention is preferably a fusion protein of a polypeptide having the amino acid sequence of SEQ ID NO: 4 and a discriminating peptide, and most preferably the above-mentioned substance of the present invention. .

The sugar chain synthesizing agent of the present invention can be used as a reagent for transferring glucuronic acid to an N-acetylgalactosamine residue present at the non-reducing end of the chondroitin skeleton. Further, since the amino acid sequence of SEQ ID NO: 4 which is a portion having the desired enzymatic activity constituting the sugar chain synthesizing agent of the present invention is a human-derived amino acid sequence, even if the sugar chain synthesizing agent of the present invention is administered to a human, it is an antigen. Is expected to have low activity, and that even if cultured cartilage or the like is transplanted to a living body after being modified with the sugar chain synthesizing agent of the present invention, no antigen-antibody reaction or inflammation caused by the sugar chain synthesizing agent of the present invention is induced. There is expected.

[0040]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 <1> Preparation of vector for expressing human GlcAT A pBluescriptII SK (+) plasmid (Kazusa DNA Research Institute: KIAA1402) containing the cDNA of GenBank Accession No. AB037823 (consisting of the nucleotide sequence of SEQ ID NO: 1) was prepared. It was digested with restriction enzymes NarI and BamHI according to a conventional method. This digestion product was subjected to agarose gel electrophoresis and the 5.4 kbp band was separated by Ge
l Extraction Kit (manufactured by QIAGEN) was used for recovery. The recovered digestion product was blunted using TaKaRa Blunting Kit (Takara Shuzo Co., Ltd.), and then HindIII linker (Takara Shuzo Co., Ltd.) was bound thereto for self-ligation.

The DNA fragment thus prepared was digested with restriction enzymes HindIII and NotI, and the digested product was subjected to agarose gel electrophoresis to recover a 2.3 kbp fragment. This DNA fragment was ligated to pFLAG-CMV-3 digested with restriction enzymes HindIII and NotI according to a conventional method. The base sequence of the obtained plasmid was sequenced according to a conventional method, and the GlcAT cDNA consisting of the base sequence of SEQ ID NO: 3 was FLA.
The vector constructed so as to be expressed as a fusion protein with the G protein was designated as pFLAGGlcAT.

<2> Preparation of Recombinant As host cells for preparing the recombinant, monkey kidney-derived cultured cells COS-7 cells (Dulbecco's adjusted minimum medium:
Hereinafter, also referred to as "DMEM") was used. Using the above-prepared pFLAGGlcAT (2 μg), TransFast (trade name: manufactured by Promega Co.) was analyzed according to the attached protocol.
It was introduced into 7 cells (cultured cells derived from monkey kidney), and transiently expressed cells were collected by a conventional method (transient: K
2) By adding antibiotic G418 at a concentration of 600 μg / ml to the medium, drug resistance screening was performed to recover stable strains (stable: K2-2 to K2-21).

The obtained culture of each clone was separated into cells and culture supernatant by centrifugation, and the anti-FLAG antibody affinity gel (M ₂ affinity gel: Sigma) was separated from the cell lysate and the culture supernatant. ) Is used to collect the fusion protein, and the anti-FLAG antibody (M ₂ : manufactured by Sigma) is used as a primary antibody and the secondary antibody is a peroxidase-labeled anti-mouse IgG antibody (manufactured by Zymed) as a Western blotting method (To.
wbin et al., PNAS USA, (1979), 76: 4350-4354) (Fig. 1). As a result, it was revealed that the K2-5 cell line expresses a fusion protein of FLAG protein and GlcAT in the medium at a particularly high concentration.

<3> Purification of fusion protein of FLAG protein and GlcAT The K2-5 strain obtained above was used in a 150 mm culture dish to prepare 20 ml of D
MEM (10% fetal bovine serum, 100 U / ml penicillin G potassium, 10
After culture at 0 μg / ml streptomycin sulfate and 600 μg / ml G418) to reach confluence, the medium was exchanged and the culture supernatant was obtained by further culturing for 3 days.

[0045] 1 mol / l Tris-HCl, pH7.
4. Glycerol and glycerol were added to final concentrations of 50 mmol / l and 20%, respectively, and stored at -80 ° C until use. After thawing the stored medium, Anti-FLAG M2-Agarose Affinity Gel (S
(IGMA) was added and mixed by inversion at 4 ° C. overnight. After the reaction, the gel was washed 3 times with Tris-buffered physiological saline (hereinafter also referred to as "TBS") / 0.05% Tween20 (trade name: manufactured by ICN), then once with TBS, and then injected with 27G. The fusion protein was purified by removing excess washing solution using a syringe with a needle.

<4> Activity measurement of purified fusion protein (1) N-acetylgalactosamine transferase (GalNAcT)
Measurement of activity 10 mmol / l MnCl ₂ , 171 μmol / l adenosine triphosphate (A
50 mmol / l MES buffer (pH 6.
To 5), 10 ml of the culture supernatant was added to the fusion protein obtained in the above <3>, and tritium-labeled UDP (uridine diphosphate) -GalNAc (9 × 10 ⁵ as an N-acetylgalactosamine residue donor was added. cpm: manufactured by NEN) and chondroitin (0.28 μg: manufactured by Seikagaku Corporation) as a galactosamine residue receptor were added, and the total amount was adjusted to 100 μl with distilled water. This reaction solution was reacted at 37 ° C for 3 hours. Then, the enzyme was inactivated by heating at 100 ° C. for 5 minutes to stop the reaction. After the reaction solution was filtered through a microfilter (Millipore) with a pore size of 0.22 μm, the flow rate was 0.
Superdex pept with 0.2 mol / l NaCl as mobile phase at 5 ml / min
ide column (30 × 10mm: made by Pharmacia)
The eluate was collected as fractions every 0.5 ml. Then, the radioactivity of each fraction was counted by a scintillation counter (Fig. 2). As a positive control, a chondroitin synthase preparation derived from Escherichia coli K4 strain (J. Biol. Chem., 277 (20
02), 21567-21575: given by Mr. Ninomiya, Seikagaku Corporation) 3 μU was used. As a result, while the positive subject transferred N-acetylgalactosamine residues to chondroitin, the fusion protein of the present invention revealed that it did not transfer N-acetylgalactosamine residues to chondroitin. became.

Further, the same measurement was carried out using chondroitin sulfate hexasaccharide (1 nmol: gifted from Mr. Tawada, Seikagaku Corporation) purified by limited decomposition with testicular hyaluronidase (FIG. 3). As a result, it was revealed that the fusion protein of the present invention does not transfer N-acetylgalactosamine residues to such oligosaccharides.

(2) Measurement of glucuronosyltransferase (GlcAT) activity In 50 mmol / l sodium acetate buffer (pH 5.6) containing 10 mmol / l MnCl ₂ , the above <3> corresponding to 10 ml of culture supernatant. The obtained fusion protein was added, and UDP-GlcA labeled with ¹⁴ C as a glucuronic acid residue donor (9 × 10 ⁵ cpm: manufactured by ICN), chondroitin (0.28 μg: biochemical) as a glucuronic acid residue acceptor. (Manufactured by Kogyo Co., Ltd.) was added, and the total amount was adjusted to 100 μl with distilled water. This reaction solution
The reaction was carried out at 37 ° C for 3 hours. Then, the enzyme was inactivated by heating at 100 ° C. for 5 minutes to stop the reaction. Microfilter with a pore size of 0.22 μm (Millipore)
After filtration through a column, it is passed through a Superdex peptide column (30 x 10 mm: Pharmacia) with a mobile phase of 0.2 mol / l NaCl at a flow rate of 0.5 ml / min, and the eluate is collected as 0.5 ml fractions. did. Then, the radioactivity of each fraction was counted by a scintillation counter (Fig. 4). In addition, as a positive target, a chondroitin synthase preparation (J.
Biol. Chem., 277 (2002), 21567-21575: gifted from Mr. Ninomiya, Seikagaku Corporation) 3 μU was used. As a result, it was revealed that the glucuronic acid residue-transferring activity to chondroitin was observed in the positive subjects, whereas the fusion protein of the present invention did not transfer the glucuronic acid residue to chondroitin. It was

Further, chondroitin sulfate hexasaccharide (1 nmol: bestowed by Mr. Tawada, Seikagaku Corporation) purified by limited decomposition with testicular hyaluronidase was added to the chondroitin synthase (J. Biol.
Chem., 277 (2002), 21567-21575: Seikagaku Kogyo Co., Ltd., presented by Mr. Ninomiya. Similar measurements were carried out using chondroitin sulfate 7-sugar prepared by transferring N-acetylgalactosamine (Fig. 5). As a result, it was revealed that the fusion protein of the present invention transfers a glucuronic acid residue to such an oligosaccharide to obtain a chondroitin sulfate octasaccharide.

The chondroitin sulfate 7-sugar as the above-mentioned acceptor is 20 mmol / l of MnCl ₂ , 0.1 mol / l of (NH ₄ ) ₂ SO ₄ , 1
50 mmol / l Tris-HCl containing mol / l ethylene glycol
Chondroitin sulfate hexasaccharide (50 μg: Mr. Tawada, Seikagaku Corporation) using 12 μU of chondroitin synthase derived from Escherichia coli K4 strain as a buffer (pH 7.2) and limited decomposition by testis hyaluronidase as N-acetylgalactosamine receptor More favorable) was used. In this reaction, 1.5 mmo as N-acetylgalactosamine donor
l / l UDP-GalNAc (manufactured by Sigma) was added, and the total amount was adjusted to 500 μl with distilled water. This reaction solution was reacted at 30 ° C for 1 hour and then heated at 100 ° C for 5 minutes to inactivate the enzyme to stop the reaction. Flow the reaction mixture at a flow rate of 2 ml / min.
Superdex 30 column with mobile phase of 2 mol / l NH ₄ HCO ₃ (60
X16 mm: manufactured by Pharmacia) and the eluate is 215 nm
It was fractionated every 2 ml while being monitored by. Fractions corresponding to chondroitin sulfate 7-sugar were pooled, lyophilized, and
It was prepared by dissolving in 1 l of distilled water.

Furthermore, 10 mmol of the fusion protein of the present invention is added.
chelate Mn ^{2+ in the} presence of 1 / l EDTA
When AT activity was measured, it became clear that the activity was completely lost (FIG. 6). This indicates that at least the fusion protein of the present invention requires a divalent cation for the reaction.

(3) Analysis of reaction product of fusion protein of the present invention In order to analyze the structure of chondroitin sulfate octasaccharide obtained by reacting chondroitin sulfate 7-saccharide with the fusion protein of the present invention in (2), The produced octasaccharide was digested with chondroitinase ABC to analyze what kind of digestion product was obtained.

That is, the radioactivity of the above octasaccharide was observed 2
The 3rd to 27th fractions were collected and lyophilized, and then desalted with a desalting column (Fast Desalting Column: Pharmacia) using 0.2 mol / l NH ₄ HCO ₃ as a mobile phase at a flow rate of 1 ml / min, Further, the eluate was divided into two and freeze-dried. On the other hand (control group), 0.1 mol / l Tr containing 30 mmol / l sodium acetate
It is dissolved in 200 μl of is-HCl buffer (pH 8.0), while the other (test group) is 100 mU of chondroitinase ABC (manufactured by Seikagaku Corporation), 0.1 mol / l containing 30 mmol / l sodium acetate.
It was dissolved in 200 μl of Tris-HCl buffer (pH 8.0). 37 each
The reaction was allowed to proceed at 30 ° C. for 30 minutes and then heated at 100 ° C. for 5 minutes to inactivate chondroitinase ABC and stop the enzymatic reaction.
The reaction solution was filtered through a microfilter (made by Millipore) having a pore size of 0.22 μm, and the flow rate was 0.5 ml / min of 0.2 mol.
Superdex peptide column (30 x 1 with / l NaCl as mobile phase)
(0 mm: Pharmacia) to elute with 0.5 ml of eluate
It fractionated every time. Then, the radioactivity of each fraction was counted by a scintillation counter (Fig. 7).

As a result, the control group was chondroitinase AB.
It is eluted with the same pattern as the original state without digestion with C, but in the test group, the peak shifts to almost the same position as Δdi-0S (a disaccharide with unsaturated group consisting of unsaturated uronic acid-N-acetylgalactosamine) did. From this fact, the fusion protein of the present invention has a non-reducing end of the chondroitin skeleton.
It was revealed that the glucuronic acid residue was transferred by β1,3 bond to N-acetylgalactosamine residue.

(4) Change in enzymatic reaction depending on substrate chain length Chondroitin sulfate derived from shark (chondroitin sulfate C:
Seikagaku Corporation and chondroitin (the above shark-derived chondroitin sulfate is chemically desulfated):
Seikagaku Corporation) and using J. Biochem., 11
1 (1992), pp.91-98, these were decomposed and purified to obtain pentasaccharide, heptose, heptose, 9-sugar, 11-sugar, and 13-sugar, respectively.
Oligosaccharides composed of sugars were prepared (these oligosaccharides derived from chondroitin sulfate were CS5, CS7, CS9, CS11,
And CS13, and similarly, these oligosaccharides derived from chondroitin are denoted as CH5, CH7, CH9, CH11, and CH13, respectively).

Using these as glucuronic acid residue receptors, the "function as glucuronic acid residue acceptor" of each oligosaccharide was measured in the same manner as in (2) (Table 1). As a result, it was revealed that oligosaccharides having 9 or more sugars are particularly preferable as the glucuronic acid residue receptor.

[0057]

[Table 1]

Under the above condition (2), changes in GlcAT activity were measured when the CS11 concentration was changed and when the UDP-GlcUA concentration was changed (when the CS11 concentration was changed. : Fig. 8, when the concentration of UDP-GlcUA was changed: Fig. 9). As a result, it became clear that the reaction rate tended to be saturated with increasing concentration. Using these, the Michaelis constant (Km) value for CS11 and UDP-GlcUA was calculated. The Km value for CS11 was 65.3 μmol / l, UDP-Glc
The Km value of UA was 82.4 μmol / l. As far as this value is seen, it is clear that the substance of the present invention has a very high affinity for the substrate.

<5> Human Normal Tissue and Cell Line
Quantification of GlcAT expression The expression level of the GlcAT gene was quantified by quantitative polymerase chain reaction (PCR) using human normal tissue and cell line cDNA. Marathon Ready cDNA (manufactured by Clontech) was used as the cDNA. For quantitative PCR of GlcAT, K2-Fw (described in SEQ ID NO: 5) and K2-Rv (described in SEQ ID NO: 6) were used as primers, and K2-MGB (a minor groove binder of Applied Biosystems) was used as a probe ( Sequence number 7 description) was used. Universal PCR MasterMix (manufactured by Applied Biosystems) is used as an enzyme and a reaction solution, and ABI PRISM 770 Sequence is used.
Quantitation was performed using a detection system (manufactured by Applied Biosystems) with a reaction solution volume of 25 μl. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a standard gene for quantification, and a standard curve for quantification was prepared using a template DNA of known concentration to standardize the expression level of the gene. Reaction temperature is 50 ℃ for 2 minutes, 95 ℃ for 10 minutes, then 95 ℃ for 15 seconds ・ 6
50 cycles of 1 minute at 0 ° C were performed (Fig. 10). As a result, GlcA
Although expression of T was observed in almost all healthy tissues and cell lines, it was revealed that T was highly expressed in placenta, small intestine, pancreas and spleen, particularly placenta.

[0060]

INDUSTRIAL APPLICABILITY According to the present invention, among enzymes involved in the synthesis of chondroitin skeleton, an enzyme having substantially no N-acetylgalactosamine transferase activity and having glucuronosyltransferase activity can be obtained by genetic engineering. Fusion proteins and their manufacture are provided.

[0061]

[Sequence list]                                SEQUENCE LISTING        <110> Kazusa DNA Research Institute       Seikagaku Corporation <120> A fusion protein having glucronic acid transferase activity <140> <141> <160> 7 <210> 1 <211> 3970 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1466) .. (3832) <400> 1 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 attga tcc ttg agg ctg tgc ccc tgg ggc acc 1492                             Ser Leu Arg Leu Cys Pro Trp Gly Thr                               1 5 cac ctg gca ggg cct acc acc atg cga ctg agc tcc ctg ttg gct ctg 1540 His Leu Ala Gly Pro Thr Thr Met Arg Leu Ser Ser Leu Leu Ala Leu  10 15 20 25 ctg cgg cca gcg ctt ccc ctc atc tta ggg ctg tct ctg ggg tgc agc 1588 Leu Arg Pro Ala Leu Pro Leu Ile Leu Gly Leu Ser Leu Gly Cys Ser                  30 35 40 ctg agc ctc ctg cgg gtt tcc tgg atc cag ggg gag gga gaa gat ccc 1636 Leu Ser Leu Leu Arg Val Ser Trp Ile Gln Gly Glu Gly Glu Asp Pro              45 50 55 tgt gtc gag gct gta ggg gag cga gga ggg cca cag aat cca gat tcc 1684 Cys Val Glu Ala Val Gly Glu Arg Gly Gly Pro Gln Asn Pro Asp Ser          60 65 70 aga gct cgg cta gac caa agt gat gaa gac ttc aaa ccc cgg att gtc 1732 Arg Ala Arg Leu Asp Gln Ser Asp Glu Asp Phe Lys Pro Arg Ile Val      75 80 85 ccc tac tac agg gac ccc aac aag ccc tac aag aag gtg ctc agg act 1780 Pro Tyr Tyr Arg Asp Pro Asn Lys Pro Tyr Lys Lys Val Leu Arg Thr  90 95 100 105 cgg tac atc cag aca gag ctg ggc tcc cgt gag cgg ttg ctg gtg gct 1828 Arg Tyr Ile Gln Thr Glu Leu Gly Ser Arg Glu Arg Leu Leu Val Ala                 110 115 120 gtc ctg acc tcc cga gct aca ctg tcc act ttg gcc gtg gct gtg aac 1876 Val Leu Thr Ser Arg Ala Thr Leu Ser Thr Leu Ala Val Ala Val Asn             125 130 135 cgt acg gtg gcc cat cac ttc cct cgg tta ctc tac ttc act ggg cag 1924 Arg Thr Val Ala His His Phe Pro Arg Leu Leu Tyr Phe Thr Gly Gln         140 145 150 cgg ggg gcc cgg gct cca gca ggg atg cag gtg gtg tct cat ggg gat 1972 Arg Gly Ala Arg Ala Pro Ala Gly Met Gln Val Val Ser His Gly Asp     155 160 165 gag cgg ccc gcc tgg ctc atg tca gag acc ctg cgc cac ctt cac aca 2020 Glu Arg Pro Ala Trp Leu Met Ser Glu Thr Leu Arg His Leu His Thr 170 175 180 185 cac ttt ggg gcc gac tac gac tgg ttc ttc atc atg cag gat gac aca 2068 His Phe Gly Ala Asp Tyr Asp Trp Phe Phe Ile Met Gln Asp Asp Thr                 190 195 200 tat gtg cag gcc ccc cgc ctg gca gcc ctt gct ggc cac ctc agc atc 2116 Tyr Val Gln Ala Pro Arg Leu Ala Ala Leu Ala Gly His Leu Ser Ile             205 210 215 aac caa gac ctg tac tta ggc cgg gca gag gag ttc att ggc gca ggc 2164 Asn Gln Asp Leu Tyr Leu Gly Arg Ala Glu Glu Phe Ile Gly Ala Gly         220 225 230 gag cag gcc cgg tac tgt cat ggg ggc ttt ggc tac ctg ttg tca cgg 2212 Glu Gln Ala Arg Tyr Cys His Gly Gly Phe Gly Tyr Leu Leu Ser Arg     235 240 245 agt ctc ctg ctt cgt ctg cgg cca cat ctg gat ggc tgc cga gga gac 2260 Ser Leu Leu Leu Arg Leu Arg Pro His Leu Asp Gly Cys Arg Gly Asp 250 255 260 265 att ctc agt gcc cgt cct gac gag tgg ctt gga cgc tgc ctc att gac 2308 Ile Leu Ser Ala Arg Pro Asp Glu Trp Leu Gly Arg Cys Leu Ile Asp                 270 275 280 tct ctg ggc gtc ggc tgt gtc tca cag cac cag ggg cag cag tat cgc 2356 Ser Leu Gly Val Gly Cys Val Ser Gln His Gln Gly Gln Gln Tyr Arg             285 290 295 tca ttt gaa ctg gcc aaa aat agg gac cct gag aag gaa ggg agc tcg 2404 Ser Phe Glu Leu Ala Lys Asn Arg Asp Pro Glu Lys Glu Gly Ser Ser         300 305 310 gct ttc ctg agt gcc ttc gcc gtg cac cct gtc tcc gaa ggt acc ctc 2452 Ala Phe Leu Ser Ala Phe Ala Val His Pro Val Ser Glu Gly Thr Leu     315 320 325 atg tac cgg ctc cac aaa cgc ttc agc gct ctg gag ttg gag cgg gct 2500 Met Tyr Arg Leu His Lys Arg Phe Ser Ala Leu Glu Leu Glu Arg Ala 330 335 340 345 tac agt gaa ata gaa caa ctg cag gct cag atc cgg aac ctg acc gtg 2548 Tyr Ser Glu Ile Glu Gln Leu Gln Ala Gln Ile Arg Asn Leu Thr Val                 350 355 360 ctg acc ccc gaa ggg gag gca ggg ctg agc tgg ccc gtt ggg ctc cct 2596 Leu Thr Pro Glu Gly Glu Ala Gly Leu Ser Trp Pro Val Gly Leu Pro             365 370 375 gct cct ttc aca cca cac tct cgc ttt gag gtg ctg ggc tgg gac tac 2644 Ala Pro Phe Thr Pro His Ser Arg Phe Glu Val Leu Gly Trp Asp Tyr         380 385 390 ttc aca gag cag cac acc ttc tcc tgt gca gat ggg gct ccc aag tgc 2692 Phe Thr Glu Gln His Thr Phe Ser Cys Ala Asp Gly Ala Pro Lys Cys     395 400 405 cca cta cag ggg gct agc agg gcg gac gtg ggt gat gcg ttg gag act 2740 Pro Leu Gln Gly Ala Ser Arg Ala Asp Val Gly Asp Ala Leu Glu Thr 410 415 420 425 gcc ctg gag cag ctc aat cgg cgc tat cag ccc cgc ctg cgc ttc cag 2788 Ala Leu Glu Gln Leu Asn Arg Arg Tyr Gln Pro Arg Leu Arg Phe Gln                 430 435 440 aag cag cga ctg ctc aac ggc tat cgg cgc ttc gac cca gca cgg ggc 2836 Lys Gln Arg Leu Leu Asn Gly Tyr Arg Arg Phe Asp Pro Ala Arg Gly             445 450 455 atg gag tac acc ctg gac ctg ctg ttg gaa tgt gtg aca cag cgt ggg 2884 Met Glu Tyr Thr Leu Asp Leu Leu Leu Glu Cys Val Thr Gln Arg Gly         460 465 470 cac cgg cgg gcc ctg gct cgc agg gtc agc ctg ctg cgg cca ctg agc 2932 His Arg Arg Ala Leu Ala Arg Arg Val Ser Leu Leu Arg Pro Leu Ser     475 480 485 cgg gtg gaa atc cta cct atg ccc tat gtc act gag gcc acc cga gtg 2980 Arg Val Glu Ile Leu Pro Met Pro Tyr Val Thr Glu Ala Thr Arg Val 490 495 500 505 cag ctg gtg ctg cca ctc ctg gtg gct gaa gct gct gca gcc ccg gct 3028 Gln Leu Val Leu Pro Leu Leu Val Ala Glu Ala Ala Ala Ala Pro Ala                 510 515 520 ttc ctc gag gcc ttt gca gcc aat gtc ctg gag cca cga gaa cat gca 3076 Phe Leu Glu Ala Phe Ala Ala Asn Val Leu Glu Pro Arg Glu His Ala             525 530 535 ttg ctc acc ctg ttg ctg gtc tac ggg cca cga gaa ggt ggc cgt gga 3124 Leu Leu Thr Leu Leu Leu Val Tyr Gly Pro Arg Glu Gly Gly Arg Gly         540 545 550 gct cca gac cca ttt ctt ggg gtg aag gct gca gca gcg gag tta gag 3172 Ala Pro Asp Pro Phe Leu Gly Val Lys Ala Ala Ala Ala Glu Leu Glu     555 560 565 cga cgg tac cct ggg acg agg ctg gcc tgg ctc gct gtg cga gca gag 3220 Arg Arg Tyr Pro Gly Thr Arg Leu Ala Trp Leu Ala Val Arg Ala Glu 570 575 580 585 gcc cct tcc cag gtg cga ctc atg gac gtg gtc tcg aag aag cac cct 3268 Ala Pro Ser Gln Val Arg Leu Met Asp Val Val Ser Lys Lys His Pro                 590 595 600 gtg gac act ctc ttc ttc ctt acc acc gtg tgg aca agg cct ggg ccc 3316 Val Asp Thr Leu Phe Phe Leu Thr Thr Val Trp Thr Arg Pro Gly Pro             605 610 615 gaa gtc ctc aac cgc tgt cgc atg aat gcc atc tct ggc tgg cag gcc 3364 Glu Val Leu Asn Arg Cys Arg Met Asn Ala Ile Ser Gly Trp Gln Ala         620 625 630 ttc ttt cca gtc cat ttc cag gag ttc aat cct gcc ctg tca cca cag 3412 Phe Phe Pro Val His Phe Gln Glu Phe Asn Pro Ala Leu Ser Pro Gln     635 640 645 aga tca ccc cca ggg ccc ccg ggg gct ggc cct gac ccc ccc tcc cct 3460 Arg Ser Pro Pro Gly Pro Pro Gly Ala Gly Pro Asp Pro Pro Ser Pro 650 655 660 665 cct ggt gct gac ccc tcc cgg ggg gct cct ata ggg ggg aga ttt gac 3508 Pro Gly Ala Asp Pro Ser Arg Gly Ala Pro Ile Gly Gly Arg Phe Asp                 670 675 680 cgg cag gct tct gcg gag ggc tgc ttc tac aac gct gac tac ctg gcg 3556 Arg Gln Ala Ser Ala Glu Gly Cys Phe Tyr Asn Ala Asp Tyr Leu Ala             685 690 695 gcc cga gcc cgg ctg gca ggt gaa ctg gca ggc cag gaa gag gag gaa 3604 Ala Arg Ala Arg Leu Ala Gly Glu Leu Ala Gly Gln Glu Glu Glu Glu         700 705 710 gcc ctg gag ggg ctg gag gtg atg gat gtt ttc ctc cgg ttc tca ggg 3652 Ala Leu Glu Gly Leu Glu Val Met Asp Val Phe Leu Arg Phe Ser Gly     715 720 725 ctc cac ctc ttt cgg gcc gta gag cca ggg ctg gtg cag aag ttc tcc 3700 Leu His Leu Phe Arg Ala Val Glu Pro Gly Leu Val Gln Lys Phe Ser 730 735 740 745 ctg cga gac tgc agc cca cgg ctc agt gaa gaa ctc tac cac cgc tgc 3748 Leu Arg Asp Cys Ser Pro Arg Leu Ser Glu Glu Leu Tyr His Arg Cys                 750 755 760 cgc ctc agc aac ctg gag ggg cta ggg ggc cgt gcc cag ctg gct atg 3796 Arg Leu Ser Asn Leu Glu Gly Leu Gly Gly Arg Ala Gln Leu Ala Met             765 770 775 gct ctc ttt gag cag gag cag gcc aat agc act tag cccgcctggg 3842 Ala Leu Phe Glu Gln Glu Gln Ala Asn Ser Thr         780 785 ggccctaacc tcattacctt tcctttgtct gcctcagccc caggaagggc aaggcaagat 3902 ggtggacaga tagagaattg ttgctgtatt ttttaaatat gaaaatgtta ttaaacatgt 3962 cttctgcc 3970 <210> 2 <211> 788 <212> PRT <213> Homo sapiens <400> 2 Ser Leu Arg Leu Cys Pro Trp Gly Thr His Leu Ala Gly Pro Thr Thr   1 5 10 15 Met Arg Leu Ser Ser Leu Leu Ala Leu Leu Arg Pro Ala Leu Pro Leu              20 25 30 Ile Leu Gly Leu Ser Leu Gly Cys Ser Leu Ser Leu Leu Arg Val Ser          35 40 45 Trp Ile Gln Gly Glu Gly Glu Asp Pro Cys Val Glu Ala Val Gly Glu      50 55 60 Arg Gly Gly Pro Gln Asn Pro Asp Ser Arg Ala Arg Leu Asp Gln Ser  65 70 75 80 Asp Glu Asp Phe Lys Pro Arg Ile Val Pro Tyr Tyr Arg Asp Pro Asn                  85 90 95 Lys Pro Tyr Lys Lys Val Leu Arg Thr Arg Tyr Ile Gln Thr Glu Leu             100 105 110 Gly Ser Arg Glu Arg Leu Leu Val Ala Val Leu Thr Ser Arg Ala Thr         115 120 125 Leu Ser Thr Leu Ala Val Ala Val Asn Arg Thr Val Ala His His Phe     130 135 140 Pro Arg Leu Leu Tyr Phe Thr Gly Gln Arg Gly Ala Arg Ala Pro Ala 145 150 155 160 Gly Met Gln Val Val Ser His Gly Asp Glu Arg Pro Ala Trp Leu Met                 165 170 175 Ser Glu Thr Leu Arg His Leu His Thr His Phe Gly Ala Asp Tyr Asp             180 185 190 Trp Phe Phe Ile Met Gln Asp Asp Thr Tyr Val Gln Ala Pro Arg Leu         195 200 205 Ala Ala Leu Ala Gly His Leu Ser Ile Asn Gln Asp Leu Tyr Leu Gly     210 215 220 Arg Ala Glu Glu Phe Ile Gly Ala Gly Glu Gln Ala Arg Tyr Cys His 225 230 235 240 Gly Gly Phe Gly Tyr Leu Leu Seru Arg Ser Leu Leu Leu Arg Leu Arg                 245 250 255 Pro His Leu Asp Gly Cys Arg Gly Asp Ile Leu Ser Ala Arg Pro Asp             260 265 270 Glu Trp Leu Gly Arg Cys Leu Ile Asp Ser Leu Gly Val Gly Cys Val         275 280 285 Ser Gln His Gln Gly Gln Gln Tyr Arg Ser Phe Glu Leu Ala Lys Asn     290 295 300 Arg Asp Pro Glu Lys Glu Gly Ser Ser Ala Phe Leu Ser Ala Phe Ala 305 310 315 320 Val His Pro Val Ser Glu Gly Thr Leu Met Tyr Arg Leu His Lys Arg                 325 330 335 Phe Ser Ala Leu Glu Leu Glu Arg Ala Tyr Ser Glu Ile Glu Gln Leu             340 345 350 Gln Ala Gln Ile Arg Asn Leu Thr Val Leu Thr Pro Glu Gly Glu Ala         355 360 365 Gly Leu Ser Trp Pro Val Gly Leu Pro Ala Pro Phe Thr Pro His Ser     370 375 380 Arg Phe Glu Val Leu Gly Trp Asp Tyr Phe Thr Glu Gln His Thr Phe 385 390 395 400 Ser Cys Ala Asp Gly Ala Pro Lys Cys Pro Leu Gln Gly Ala Ser Arg                 405 410 415 Ala Asp Val Gly Asp Ala Leu Glu Thr Ala Leu Glu Gln Leu Asn Arg             420 425 430 Arg Tyr Gln Pro Arg Leu Arg Phe Gln Lys Gln Arg Leu Leu Asn Gly         435 440 445 Tyr Arg Arg Phe Asp Pro Ala Arg Gly Met Glu Tyr Thr Leu Asp Leu     450 455 460 Leu Leu Glu Cys Val Thr Gln Arg Gly His Arg Arg Ala Leu Ala Arg 465 470 475 480 Arg Val Ser Leu Leu Arg Pro Leu Ser Arg Val Glu Ile Leu Pro Met                 485 490 495 Pro Tyr Val Thr Glu Ala Thr Arg Val Gln Leu Val Leu Pro Leu Leu             500 505 510 Val Ala Glu Ala Ala Ala Ala Pro Ala Phe Leu Glu Ala Phe Ala Ala         515 520 525 Asn Val Leu Glu Pro Arg Glu His Ala Leu Leu Thr Leu Leu Leu Val     530 535 540 Tyr Gly Pro Arg Glu Gly Gly Arg Gly Ala Pro Asp Pro Phe Leu Gly 545 550 555 560 Val Lys Ala Ala Ala Ala Glu Leu Glu Arg Arg Tyr Pro Gly Thr Arg                 565 570 575 Leu Ala Trp Leu Ala Val Arg Ala Glu Ala Pro Ser Gln Val Arg Leu             580 585 590 Met Asp Val Val Ser Lys Lys His Pro Val Asp Thr Leu Phe Phe Leu         595 600 605 Thr Thr Val Trp Thr Arg Pro Gly Pro Glu Val Leu Asn Arg Cys Arg     610 615 620 Met Asn Ala Ile Ser Gly Trp Gln Ala Phe Phe Pro Val His Phe Gln 625 630 635 640 Glu Phe Asn Pro Ala Leu Ser Pro Gln Arg Ser Pro Pro Gly Pro Pro                 645 650 655 Gly Ala Gly Pro Asp Pro Pro Ser Pro Pro Gly Ala Asp Pro Ser Arg             660 665 670 Gly Ala Pro Ile Gly Gly Arg Phe Asp Arg Gln Ala Ser Ala Glu Gly         675 680 685 Cys Phe Tyr Asn Ala Asp Tyr Leu Ala Ala Arg Ala Arg Leu Ala Gly     690 695 700 Glu Leu Ala Gly Gln Glu Glu Glu Glu Ala Leu Glu Gly Leu Glu Val 705 710 715 720 Met Asp Val Phe Leu Arg Phe Ser Gly Leu His Leu Phe Arg Ala Val                 725 730 735 Glu Pro Gly Leu Val Gln Lys Phe Ser Leu Arg Asp Cys Ser Pro Arg             740 745 750 Leu Ser Glu Glu Leu Tyr His Arg Cys Arg Leu Ser Asn Leu Glu Gly         755 760 765 Leu Gly Gly Arg Ala Gln Leu Ala Met Ala Leu Phe Glu Gln Glu Gln     770 775 780 Ala Asn Ser Thr 785 <210> 3 <211> 2222 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: fragment from       cDNA of KIAA1402 <220> <221> CDS <222> (3) .. (2222) <400> 3 gg atc cag ggg gag gga gaa gat ccc tgt gtc gag gct gta ggg gag 47    Ile Gln Gly Glu Gly Glu Asp Pro Cys Val Glu Ala Val Gly Glu      1 5 10 15 cga gga ggg cca cag aat cca gat tcc aga gct cgg cta gac caa agt 95 Arg Gly Gly Pro Gln Asn Pro Asp Ser Arg Ala Arg Leu Asp Gln Ser                  20 25 30 gat gaa gac ttc aaa ccc cgg att gtc ccc tac tac agg gac ccc aac 143 Asp Glu Asp Phe Lys Pro Arg Ile Val Pro Tyr Tyr Arg Asp Pro Asn              35 40 45 aag ccc tac aag aag gtg ctc agg act cgg tac atc cag aca gag ctg 191 Lys Pro Tyr Lys Lys Val Leu Arg Thr Arg Tyr Ile Gln Thr Glu Leu          50 55 60 ggc tcc cgt gag cgg ttg ctg gtg gct gtc ctg acc tcc cga gct aca 239 Gly Ser Arg Glu Arg Leu Leu Val Ala Val Leu Thr Ser Arg Ala Thr      65 70 75 ctg tcc act ttg gcc gtg gct gtg aac cgt acg gtg gcc cat cac ttc 287 Leu Ser Thr Leu Ala Val Ala Val Asn Arg Thr Val Ala His His Phe  80 85 90 95 cct cgg tta ctc tac ttc act ggg cag cgg ggg gcc cgg gct cca gca 335 Pro Arg Leu Leu Tyr Phe Thr Gly Gln Arg Gly Ala Arg Ala Pro Ala                 100 105 110 ggg atg cag gtg gtg tct cat ggg gat gag cgg ccc gcc tgg ctc atg 383 Gly Met Gln Val Val Ser His Gly Asp Glu Arg Pro Ala Trp Leu Met             115 120 125 tca gag acc ctg cgc cac ctt cac aca cac ttt ggg gcc gac tac gac 431 Ser Glu Thr Leu Arg His Leu His Thr His Phe Gly Ala Asp Tyr Asp         130 135 140 tgg ttc ttc atc atg cag gat gac aca tat gtg cag gcc ccc cgc ctg 479 Trp Phe Phe Ile Met Gln Asp Asp Thr Tyr Val Gln Ala Pro Arg Leu     145 150 155 gca gcc ctt gct ggc cac ctc agc atc aac caa gac ctg tac tta ggc 527 Ala Ala Leu Ala Gly His Leu Ser Ile Asn Gln Asp Leu Tyr Leu Gly 160 165 170 175 cgg gca gag gag ttc att ggc gca ggc gag cag gcc cgg tac tgt cat 575 Arg Ala Glu Glu Phe Ile Gly Ala Gly Glu Gln Ala Arg Tyr Cys His                 180 185 190 ggg ggc ttt ggc tac ctg ttg tca cgg agt ctc ctg ctt cgt ctg cgg 623 Gly Gly Phe Gly Tyr Leu Leu Seru Arg Ser Leu Leu Leu Arg Leu Arg             195 200 205 cca cat ctg gat ggc tgc cga gga gac att ctc agt gcc cgt cct gac 671 Pro His Leu Asp Gly Cys Arg Gly Asp Ile Leu Ser Ala Arg Pro Asp         210 215 220 gag tgg ctt gga cgc tgc ctc att gac tct ctg ggc gtc ggc tgt gtc 719 Glu Trp Leu Gly Arg Cys Leu Ile Asp Ser Leu Gly Val Gly Cys Val     225 230 235 tca cag cac cag ggg cag cag tat cgc tca ttt gaa ctg gcc aaa aat 767 Ser Gln His Gln Gly Gln Gln Tyr Arg Ser Phe Glu Leu Ala Lys Asn 240 245 250 255 agg gac cct gag aag gaa ggg agc tcg gct ttc ctg agt gcc ttc gcc 815 Arg Asp Pro Glu Lys Glu Gly Ser Ser Ala Phe Leu Ser Ala Phe Ala                 260 265 270 gtg cac cct gtc tcc gaa ggt acc ctc atg tac cgg ctc cac aaa cgc 863 Val His Pro Val Ser Glu Gly Thr Leu Met Tyr Arg Leu His Lys Arg             275 280 285 ttc agc gct ctg gag ttg gag cgg gct tac agt gaa ata gaa caa ctg 911 Phe Ser Ala Leu Glu Leu Glu Arg Ala Tyr Ser Glu Ile Glu Gln Leu         290 295 300 cag gct cag atc cgg aac ctg acc gtg ctg acc ccc gaa ggg gag gca 959 Gln Ala Gln Ile Arg Asn Leu Thr Val Leu Thr Pro Glu Gly Glu Ala     305 310 315 ggg ctg agc tgg ccc gtt ggg ctc cct gct cct ttc aca cca cac tct 1007 Gly Leu Ser Trp Pro Val Gly Leu Pro Ala Pro Phe Thr Pro His Ser 320 325 330 335 cgc ttt gag gtg ctg ggc tgg gac tac ttc aca gag cag cac acc ttc 1055 Arg Phe Glu Val Leu Gly Trp Asp Tyr Phe Thr Glu Gln His Thr Phe                 340 345 350 tcc tgt gca gat ggg gct ccc aag tgc cca cta cag ggg gct agc agg 1103 Ser Cys Ala Asp Gly Ala Pro Lys Cys Pro Leu Gln Gly Ala Ser Arg             355 360 365 gcg gac gtg ggt gat gcg ttg gag act gcc ctg gag cag ctc aat cgg 1151 Ala Asp Val Gly Asp Ala Leu Glu Thr Ala Leu Glu Gln Leu Asn Arg         370 375 380 cgc tat cag ccc cgc ctg cgc ttc cag aag cag cga ctg ctc aac ggc 1199 Arg Tyr Gln Pro Arg Leu Arg Phe Gln Lys Gln Arg Leu Leu Asn Gly     385 390 395 tat cgg cgc ttc gac cca gca cgg ggc atg gag tac acc ctg gac ctg 1247 Tyr Arg Arg Phe Asp Pro Ala Arg Gly Met Glu Tyr Thr Leu Asp Leu 400 405 410 415 ctg ttg gaa tgt gtg aca cag cgt ggg cac cgg cgg gcc ctg gct cgc 1295 Leu Leu Glu Cys Val Thr Gln Arg Gly His Arg Arg Ala Leu Ala Arg                 420 425 430 agg gtc agc ctg ctg cgg cca ctg agc cgg gtg gaa atc cta cct atg 1343 Arg Val Ser Leu Leu Arg Pro Leu Ser Arg Val Glu Ile Leu Pro Met             435 440 445 ccc tat gtc act gag gcc acc cga gtg cag ctg gtg ctg cca ctc ctg 1391 Pro Tyr Val Thr Glu Ala Thr Arg Val Gln Leu Val Leu Pro Leu Leu         450 455 460 gtg gct gaa gct gct gca gcc ccg gct ttc ctc gag gcc ttt gca gcc 1439 Val Ala Glu Ala Ala Ala Ala Pro Ala Phe Leu Glu Ala Phe Ala Ala     465 470 475 aat gtc ctg gag cca cga gaa cat gca ttg ctc acc ctg ttg ctg gtc 1487 Asn Val Leu Glu Pro Arg Glu His Ala Leu Leu Thr Leu Leu Leu Val 480 485 490 495 tac ggg cca cga gaa ggt ggc cgt gga gct cca gac cca ttt ctt ggg 1535 Tyr Gly Pro Arg Glu Gly Gly Arg Gly Ala Pro Asp Pro Phe Leu Gly                 500 505 510 gtg aag gct gca gca gcg gag tta gag cga cgg tac cct ggg acg agg 1583 Val Lys Ala Ala Ala Ala Glu Leu Glu Arg Arg Tyr Pro Gly Thr Arg             515 520 525 ctg gcc tgg ctc gct gtg cga gca gag gcc cct tcc cag gtg cga ctc 1631 Leu Ala Trp Leu Ala Val Arg Ala Glu Ala Pro Ser Gln Val Arg Leu         530 535 540 atg gac gtg gtc tcg aag aag cac cct gtg gac act ctc ttc ttc ctt 1679 Met Asp Val Val Ser Lys Lys His Pro Val Asp Thr Leu Phe Phe Leu     545 550 555 acc acc gtg tgg aca agg cct ggg ccc gaa gtc ctc aac cgc tgt cgc 1727 Thr Thr Val Trp Thr Arg Pro Gly Pro Glu Val Leu Asn Arg Cys Arg 560 565 570 575 atg aat gcc atc tct ggc tgg cag gcc ttc ttt cca gtc cat ttc cag 1775 Met Asn Ala Ile Ser Gly Trp Gln Ala Phe Phe Pro Val His Phe Gln                 580 585 590 gag ttc aat cct gcc ctg tca cca cag aga tca ccc cca ggg ccc ccg 1823 Glu Phe Asn Pro Ala Leu Ser Pro Gln Arg Ser Pro Pro Gly Pro Pro             595 600 605 ggg gct ggc cct gac ccc ccc tcc cct cct ggt gct gac ccc tcc cgg 1871 Gly Ala Gly Pro Asp Pro Pro Ser Pro Pro Gly Ala Asp Pro Ser Arg         610 615 620 ggg gct cct ata ggg ggg aga ttt gac cgg cag gct tct gcg gag ggc 1919 Gly Ala Pro Ile Gly Gly Arg Phe Asp Arg Gln Ala Ser Ala Glu Gly     625 630 635 tgc ttc tac aac gct gac tac ctg gcg gcc cga gcc cgg ctg gca ggt 1967 Cys Phe Tyr Asn Ala Asp Tyr Leu Ala Ala Arg Ala Arg Leu Ala Gly 640 645 650 655 gaa ctg gca ggc cag gaa gag gag gaa gcc ctg gag ggg ctg gag gtg 2015 Glu Leu Ala Gly Gln Glu Glu Glu Glu Ala Leu Glu Gly Leu Glu Val                 660 665 670 atg gat gtt ttc ctc cgg ttc tca ggg ctc cac ctc ttt cgg gcc gta 2063 Met Asp Val Phe Leu Arg Phe Ser Gly Leu His Leu Phe Arg Ala Val             675 680 685 gag cca ggg ctg gtg cag aag ttc tcc ctg cga gac tgc agc cca cgg 2111 Glu Pro Gly Leu Val Gln Lys Phe Ser Leu Arg Asp Cys Ser Pro Arg         690 695 700 ctc agt gaa gaa ctc tac cac cgc tgc cgc ctc agc aac ctg gag ggg 2159 Leu Ser Glu Glu Leu Tyr His Arg Cys Arg Leu Ser Asn Leu Glu Gly     705 710 715 cta ggg ggc cgt gcc cag ctg gct atg gct ctc ttt gag cag gag cag 2207 Leu Gly Gly Arg Ala Gln Leu Ala Met Ala Leu Phe Glu Gln Glu Gln 720 725 730 735 gcc aat agc act tag 2222 Ala Asn Ser Thr                 740 <210> 4 <211> 739 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: fragment from       cDNA of KIAA1402 <400> 4 Ile Gln Gly Glu Gly Glu Asp Pro Cys Val Glu Ala Val Gly Glu Arg   1 5 10 15 Gly Gly Pro Gln Asn Pro Asp Ser Arg Ala Arg Leu Asp Gln Ser Asp              20 25 30 Glu Asp Phe Lys Pro Arg Ile Val Pro Tyr Tyr Arg Asp Pro Asn Lys          35 40 45 Pro Tyr Lys Lys Val Leu Arg Thr Arg Tyr Ile Gln Thr Glu Leu Gly      50 55 60 Ser Arg Glu Arg Leu Leu Val Ala Val Leu Thr Ser Arg Ala Thr Leu  65 70 75 80 Ser Thr Leu Ala Val Ala Val Asn Arg Thr Val Ala His His Phe Pro                  85 90 95 Arg Leu Leu Tyr Phe Thr Gly Gln Arg Gly Ala Arg Ala Pro Ala Gly             100 105 110 Met Gln Val Val Ser His Gly Asp Glu Arg Pro Ala Trp Leu Met Ser         115 120 125 Glu Thr Leu Arg His Leu His Thr His Phe Gly Ala Asp Tyr Asp Trp     130 135 140 Phe Phe Ile Met Gln Asp Asp Thr Tyr Val Gln Ala Pro Arg Leu Ala 145 150 155 160 Ala Leu Ala Gly His Leu Ser Ile Asn Gln Asp Leu Tyr Leu Gly Arg                 165 170 175 Ala Glu Glu Phe Ile Gly Ala Gly Glu Gln Ala Arg Tyr Cys His Gly             180 185 190 Gly Phe Gly Tyr Leu Leu Ser Arg Ser Leu Leu Leu Arg Leu Arg Pro         195 200 205 His Leu Asp Gly Cys Arg Gly Asp Ile Leu Ser Ala Arg Pro Asp Glu     210 215 220 Trp Leu Gly Arg Cys Leu Ile Asp Ser Leu Gly Val Gly Cys Val Ser 225 230 235 240 Gln His Gln Gly Gln Gln Tyr Arg Ser Phe Glu Leu Ala Lys Asn Arg                 245 250 255 Asp Pro Glu Lys Glu Gly Ser Ser Ala Phe Leu Ser Ala Phe Ala Val             260 265 270 His Pro Val Ser Glu Gly Thr Leu Met Tyr Arg Leu His Lys Arg Phe         275 280 285 Ser Ala Leu Glu Leu Glu Arg Ala Tyr Ser Glu Ile Glu Gln Leu Gln     290 295 300 Ala Gln Ile Arg Asn Leu Thr Val Leu Thr Pro Glu Gly Glu Ala Gly 305 310 315 320 Leu Ser Trp Pro Val Gly Leu Pro Ala Pro Phe Thr Pro His Ser Arg                 325 330 335 Phe Glu Val Leu Gly Trp Asp Tyr Phe Thr Glu Gln His Thr Phe Ser             340 345 350 Cys Ala Asp Gly Ala Pro Lys Cys Pro Leu Gln Gly Ala Ser Arg Ala         355 360 365 Asp Val Gly Asp Ala Leu Glu Thr Ala Leu Glu Gln Leu Asn Arg Arg     370 375 380 Tyr Gln Pro Arg Leu Arg Phe Gln Lys Gln Arg Leu Leu Asn Gly Tyr 385 390 395 400 Arg Arg Phe Asp Pro Ala Arg Gly Met Glu Tyr Thr Leu Asp Leu Leu                 405 410 415 Leu Glu Cys Val Thr Gln Arg Gly His Arg Arg Ala Leu Ala Arg Arg             420 425 430 Val Ser Leu Leu Arg Pro Leu Ser Arg Val Glu Ile Leu Pro Met Pro         435 440 445 Tyr Val Thr Glu Ala Thr Arg Val Gln Leu Val Leu Pro Leu Leu Val     450 455 460 Ala Glu Ala Ala Ala Ala Pro Ala Phe Leu Glu Ala Phe Ala Ala Asn 465 470 475 480 Val Leu Glu Pro Arg Glu His Ala Leu Leu Thr Leu Leu Leu Val Tyr                 485 490 495 Gly Pro Arg Glu Gly Gly Arg Gly Ala Pro Asp Pro Phe Leu Gly Val             500 505 510 Lys Ala Ala Ala Ala Glu Leu Glu Arg Arg Tyr Pro Gly Thr Arg Leu         515 520 525 Ala Trp Leu Ala Val Arg Ala Glu Ala Pro Ser Gln Val Arg Leu Met     530 535 540 Asp Val Val Ser Lys Lys His Pro Val Asp Thr Leu Phe Phe Leu Thr 545 550 555 560 Thr Val Trp Thr Arg Pro Gly Pro Glu Val Leu Asn Arg Cys Arg Met                 565 570 575 Asn Ala Ile Ser Gly Trp Gln Ala Phe Phe Pro Val His Phe Gln Glu             580 585 590 Phe Asn Pro Ala Leu Ser Pro Gln Arg Ser Pro Pro Gly Pro Pro Gly         595 600 605 Ala Gly Pro Asp Pro Pro Ser Pro Pro Gly Ala Asp Pro Ser Arg Gly     610 615 620 Ala Pro Ile Gly Gly Arg Phe Asp Arg Gln Ala Ser Ala Glu Gly Cys 625 630 635 640 Phe Tyr Asn Ala Asp Tyr Leu Ala Ala Arg Ala Arg Leu Ala Gly Glu                 645 650 655 Leu Ala Gly Gln Glu Glu Glu Glu Ala Leu Glu Gly Leu Glu Val Met             660 665 670 Asp Val Phe Leu Arg Phe Ser Gly Leu His Leu Phe Arg Ala Val Glu         675 680 685 Pro Gly Leu Val Gln Lys Phe Ser Leu Arg Asp Cys Ser Pro Arg Leu     690 695 700 Ser Glu Glu Leu Tyr His Arg Cys Arg Leu Ser Asn Leu Glu Gly Leu 705 710 715 720 Gly Gly Arg Ala Gln Leu Ala Met Ala Leu Phe Glu Gln Glu Gln Ala                 725 730 735 Asn Ser Thr <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 5'primer for       quantitative PCR method <400> 5 gtgaaataga acaactgcag gctc 24 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 3'primer for       quantitative PCR method <400> 6 gagaaggtgt gctgctctgt ga 22 <210> 7 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Probe for       quantitative PCR method <400> 7 cggaacctga ccgtgc 16

[0062]

[Brief description of drawings]

FIG. 1 is a photograph showing a culture of each transformant analyzed by Western blotting. The arrow indicates the band of the substance of the present invention. In the figure, "Transient" is a temporarily introduced strain,
"Stable" is a stably introduced strain, "Mock" is a negative control, and "K2"
~ "2-21" indicates a strain into which a gene encoding the substance of the present invention has been introduced. "Medium" indicates the culture supernatant, and "Cell l"
“Ayer” indicates cell lysate.

FIG. 2 is a diagram showing N-acetylgalactosamine transfer activity for chondroitin. The vertical axis represents N-acetylgalactosaminyltransferase activity by radioactivity per test tube, and the horizontal axis represents fraction number. White circles represent the substance of the present invention, and black circles represent the positive control.

FIG. 3 is a diagram showing N-acetylgalactosaminyltransferase activity against chondroitin sulfate hexasaccharide. The vertical axis is N-
The acetylgalactosamine transferase activity is shown by the radioactivity per test tube, and the horizontal axis shows the fraction number. White circles represent the substance of the present invention, and black circles represent the positive control.

FIG. 4 is a diagram showing glucuronosyl transfer activity for chondroitin. The vertical axis represents glucuronosyltransferase activity by radioactivity per test tube, and the horizontal axis represents fraction number. White circles represent the substance of the present invention, and black circles represent the positive control.

FIG. 5 is a graph showing glucuronic acid transfer activity for chondroitin sulfate 7-sugar. The vertical axis represents glucuronosyltransferase activity by radioactivity per test tube, and the horizontal axis represents fraction number. White circles represent the substance of the present invention, and black circles represent the positive control.

FIG. 6 shows the inhibition of glucuronic acid transfer activity by ethylenediaminetetraacetic acid. The vertical axis shows the glucuronosyltransferase activity by the radioactivity per test tube,
The horizontal axis represents the fraction number. White circles represent the substance of the present invention in the absence of ethylenediaminetetraacetic acid, black circles represent the substance of the present invention in the presence of ethylenediaminetetraacetic acid, and white squares represent a negative control.

FIG. 7 is a diagram showing chondroitin sulfate octasaccharide produced by transferring glucuronic acid to chondroitin sulfate heptsaccharide and the effect of enzymatic degradation thereof. The vertical axis represents radioactivity due to ¹⁴ C per test tube, and the horizontal axis represents fraction number. White circles represent the chondroitin sulfate octasaccharide prepared by the substance of the present invention, and black circles represent the product obtained by digesting the chondroitin sulfate octasaccharide with chondroitinase ABC.

FIG. 8 is a graph showing the effect on glucuronosyl transfer activity that appears when the concentration of chondroitin sulfate 11 sugar is changed. The vertical axis represents glucuronosyltransferase activity, and the horizontal axis represents the concentration of chondroitin sulfate 11 sugar.

FIG. 9 is a diagram showing the effect on glucuronosyl transfer activity that appears when the concentration of UDP-GlcUA is changed. The vertical axis represents glucuronosyltransferase activity, and the horizontal axis represents UDP-GlcUA concentration.

FIG. 10 is a diagram in which the expression of the enzyme of the present invention in human healthy tissues and cell lines was measured by a quantitative PCR reaction. The vertical axis represents the ratio of the glucuronosyltransferase gene expression level to the glyceraldehyde-3-phosphate dehydrogenase expression level (control), and the horizontal axis represents the test human normal tissue name and test cell line name.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C12N 1/19 C12N 1/21 1/21 9/00 5/10 9/10 9/00 C12P 19/26 9/10 C12N 15/00 ZNAA C12P 19/26 5/00 A (72) Inventor Koji Kizen 2-102-1, Meito-ku, Nagoya City, Aichi Prefecture Espasha Corporation 1201 (72) Inventor Masatoshi Goto Ibaraki 1-1-1 Umezono, Tsukuba, Japan Independent administrative agency, National Institute of Advanced Industrial Science and Technology (72) Inventor Toshinori Yada 27-2 Nishiyata-cho, Kuwana-shi, Mie F-term (reference) 4B024 AA20 BA07 BA10 BA80 CA01 CA07 DA02 EA04 GA11 HA01 HA03 4B050 CC04 CC05 DD11 FF14E LL05 4B064 AF21 CA10 CA21 CB30 CE10 DA01 4B065 AA90X AA93Y AB01 AC14 AC15 BA02 BA25 BD14 CA24 CA29 CA44 CA60 4H045 AA10 AA11 AA20 BA10 BA41 CA40 DA75 DA89 FA72EA74 GA20 FA72 FA20 GA74 FA72 FA20 GA74

Claims (13)

[Claims] 1. A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 4 or an amino acid sequence having one or more constituent amino acid substitutions, deletions, insertions or transpositions in the amino acid sequence represented by SEQ ID NO: 4, and a discriminating peptide. 1. A fusion protein which is fused and has an enzymatic activity of transferring glucuronic acid from a glucuronic acid donor to an N-acetylgalactosamine residue at the non-reducing end present in the chondroitin skeleton. 2. A non-reducing terminal from a glucuronic acid donor
N-acetylgalactosamine residue, which transfers a glucuronic acid residue to the N-acetylgalactosamine oligosaccharide consisting of 7 sugars having a chondroitin skeleton.
From the acetylgalactosamine donor, the non-reducing end is a glucuronic acid residue, and the oligosaccharide consisting of a hexasaccharide having a chondroitin skeleton is substantially
The fusion protein according to claim 1, which has no activity of transferring an N-acetylgalactosamine residue. 3. The discriminating peptide is a signal peptide, protein kinase A, protein A, glutathione S-transferase, His tag, myc tag, FLAG protein, T7 tag, S tag, HSV tag, pelB, HA tag, Trx tag, CBP. Tags, CBD tags, CBR tags, β-lac / blu, β-gal, luc, HP-Thio, HS
P, Lnγ, Fn, GFP, YFP, CFP, BFP, DsRed, DsRed2, MB
The fusion protein according to claim 1 or 2, which is any one peptide selected from the group consisting of P, LacZ, IgG, avidin, and protein G. 4. A DNA encoding the fusion protein according to claim 1. 5. A vector containing the DNA according to claim 4. 6. A transformant obtained by introducing the vector according to claim 5 into a host cell. 7. Growing the transformant according to claim 6,
The method for producing a fusion protein according to claim 1, wherein the fusion protein is isolated from the grown product. 8. An antibody which specifically recognizes the fusion protein according to any one of claims 1 to 3. 9. A glucuronosyltransferase having the following properties. (A) Action The glucuronic acid residue is transferred from the glucuronic acid donor to the acceptor N-acetylgalactosamine residue. (B) From the substrate-specific glucuronic acid donor, the non-reducing end is an N-acetylgalactosamine residue and has a chondroitin skeleton 7
It transfers a glucuronic acid residue to the N-acetylgalactosamine of the oligosaccharide consisting of sugar, but from the N-acetylgalactosamine donor, the non-reducing end is a glucuronic acid residue and consists of a hexasaccharide having a chondroitin skeleton. The oligosaccharide has substantially no activity to transfer the N-acetylgalactosamine residue to the glucuronic acid. (C) Inhibition of activity In the coexistence of ethylenediaminetetraacetic acid, it exhibits substantially no enzyme activity. 10. The amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 contains an amino acid sequence having substitution, deletion, insertion, or transposition of one or more constituent amino acids. The glucuronosyltransferase described. 11. The fusion protein according to any one of claims 1 to 3 or the glucuronic acid according to claim 9 or 10 for a sugar chain having an N-acetylgalactosamine residue at a non-reducing end and a chondroitin skeleton. A method for producing a sugar chain having a chondroitin skeleton, which comprises transferring a glucuronic acid from a glucuronic acid donor to an N-acetylgalactosamine residue at the non-reducing end of the sugar chain by causing a transferase to act. 12. A protein comprising a protein comprising an amino acid sequence of SEQ ID NO: 4 or a polypeptide consisting of an amino acid sequence having one or more constituent amino acid substitutions, deletions, insertions or transpositions in the amino acid sequence of SEQ ID NO: 4. , A sugar chain synthesizing agent having an activity of transferring glucuronic acid from a glucuronic acid donor to a sugar chain having a chondroitin skeleton having an N-acetylgalactosamine residue at a non-reducing end. 13. A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 4 or an amino acid sequence having one or more constituent amino acid substitutions, deletions, insertions or transpositions in the amino acid sequence represented by SEQ ID NO: 4, and a discriminating peptide. 13. The sugar chain synthesizing agent according to claim 12, comprising the fusion protein of.