JP4911403B2 - Glycosyltransferase containing glucose transferase - Google Patents

Description

  The present invention relates to a glycosyl transfer agent containing glucose transferase, which transfers glucose to fucose with β1,3 bonds.

  Among O-linked sugar chains, O-Fuc sugar chains in which fucose (hereinafter also referred to as “Fuc”) is bound to a serine residue in a protein are involved in various biological functions such as Notch signaling. It has been reported that There are two types of O-Fuc sugar chains, and different O-Fuc sugar chains have been reported on epidermal growth factor (hereinafter also referred to as “EGF”)-like repeat sequences and thrombospondin-like repeat sequences.

The serine residue in the EGF-like repeat sequence has a sugar chain structure represented by the following formula (1) (Nishimura, H. et al., 1992).
Sia-Gal-GlcNAc-Fuc-Ser (1)
In formula (1), “Sia” represents a sialic acid residue, “Gal” represents a galactose residue, “GlcNAc” represents an N-acetylglucosamine residue, and “Fuc” represents a serine represented by Ser. A fucose residue α-bonded to the side chain of the residue, “Ser” represents a serine residue (including a case in the peptide chain) in which the fucose residue represented by Fuc is bound to the side chain, and “− "Indicates a glycosidic bond. It is protein-O-fucose transferase 1 (hereinafter also referred to as “POFUT1”) that transfers Fuc to Ser in the structure of formula (1), and GlcNAc is transferred to Fuc by manic fringe and radicals. There are three types of β1,3N-acetylglucosaminyltransferase called fringe and lunatic fringe (Moloney, DJ et al., 2000). In the case of Notch signaling, a ligand to be bound is selected depending on whether or not N-acetylglucosamine is transferred by these fringes, and each signal transduction is caused.

On the other hand, the serine residue in the thrombospondin-like repeat sequence has a sugar chain structure represented by the following formula (2) different from the sugar chain structure in the EGF-like repeat sequence (Hofsteenge, J. et al., 2001).
Glc-Fuc-Ser (2)
In Formula (2), “Glc” represents a glucose residue, “Fuc” represents a fucose residue α-bonded to the side chain of the serine residue represented by Ser, and “Ser” is represented by Fuc. A fucose residue is a serine residue (including a case in a peptide chain) bonded to a side chain, and “-” indicates a glycosidic bond. It is protein-O-fucose transferase 2 (hereinafter also referred to as “POFUT2”) that transfers Fuc to Ser in the structure of formula (2) (Luo, Y. et al., 2003). Enzymes with activity to transfer Glc have not been isolated / purified so far. In addition, a substance having endogenous β3Glc-T activity is present in Chinese Hamster Ovary cells (CHO cells), but Fuc-α-pNp and UDP are used as an enzyme source. -It has been reported that Glcβ1,3Fuc-α-pNp is produced when reacted with Glc (Moloney, DJ et al., 1999).
JP 2004-215619 A Nishimura, H .; , T. Takao, S .; Hase, Y .; Shimonishi and S. Iwanaga, Human factor IX has a tetraacsaccharide O-glycosidically linked to serine 61 through the fucose residue. J. et al. Biol. Chem. , 267, 17520-17525 (1992) Moloney, D.M. J. et al. , V. M.M. Panin, S.M. H. Johnston, J.A. Chen, L .; Shao, R .; Wilson, Y .; Wang, P.A. Stanley, K.M. D. Irvine, R.M. S. Haltiwanger and T.W. F. Vogt, Fringe is a glycosyltransferase reference that modifies Notch. Nature, 406, 369-375 (2000) Hofsteine, J. et al. K. G. Huwiler, B.M. Macek, D.M. Hess, J .; Lawler, D.C. F. Mosher and J.M. P. Katalinic, C-mannosylation and O-fucosylation of the thrombospondin type 1 module. J. et al. Biol. Chem. , 276, 6485-6498 (2001) Luo, Y .; , W .; Vorndam, V.M. Panin, S.M. Shi, P.A. Stanley and R.C. S. Haltiwanger, Identification of a novel energy-responsible for O-fucosylation of thrombospondin type 1 repeats. Glycobiology, 13: abstract 180 (2003) Moloney, D.M. J. et al. , And R. S. Haltiwanger, The O-linked fucose glycosylation pathway: identification and charac- terization of auridine diphospholluciferase: fucose-β1, 3-glucosyltransferase. Glycobiology, 9, 679-687 (1999)

By isolating a protein having an activity of transferring glucose to fucose (hereinafter also referred to as “glucose transferase” or “Glc-T”) and clarifying the structure of the gene, the enzyme by genetic engineering techniques, etc. And the diagnosis of diseases based on the gene and the like becomes possible. However, the enzyme is not yet known, and there is no clue about the isolation of the enzyme and the identification of the gene. Therefore, an antibody against the enzyme has not been prepared.
Accordingly, an object of the present invention is to identify a protein having an activity of transferring glucose to fucose and provide a glycosyl transfer agent containing the protein or a nucleic acid encoding the protein. The protein expressed by genetic engineering can also be used for antibody production. Furthermore, the protein of the present invention and an antibody against the protein can be applied to immunoassay methods such as immunohistochemical staining, RIA and EIA.

  As described above, the target glucose transferase is not yet known. To date, the group of the present inventors has succeeded in isolating a transferase having an activity of transferring galactose to N-acetylglucosamine (Japanese Patent Application Laid-Open No. 2004-215619). Subsequently, when a partial sequence of the cDNA of this enzyme was expressed, the enzyme was found to have an activity of transferring glucose via a β1,3 bond to a receptor substrate having fucose at the non-reducing end. It came to complete.

  That is, according to the present invention, there is provided a protein (glucosyltransferase) having an activity of transferring glucose to a fucose residue through a β1,3 bond, or a nucleic acid encoding the same. As the glycosyltransferase of the present invention, glucose transferase (Glc-T) may be used directly, or a nucleic acid encoding Glc-T inserted into an appropriate expression vector, for example. Good. The Glc-T protein or nucleic acid encoding the same used for the glycosyltransferase of the present invention is preferably derived from a mammal, more preferably a human, a mouse, a yellow drosophila, a nematode, and even more preferably a human or a mouse. . In the present specification, “Glc-T” indicates a Glc-T protein, a nucleic acid encoding the protein, or both a protein and a nucleic acid unless otherwise specified.

  In one embodiment of the present invention, the Glc-T protein used in the glycosyltransferase of the present invention is a protein having the amino acid sequence represented by SEQ ID NO: 1, or one or more amino acids in the sequence are It may be a protein that has an amino acid sequence substituted or deleted, or having one or more amino acids inserted or added in the sequence. Also, such an amino acid sequence is at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90% of the amino acid sequence shown in SEQ ID NO: 1. Have identity.

  According to the present invention, there is provided a protein having an amino acid sequence in which all or part of amino acid residues 1-28 is deleted from the amino acid sequence shown in SEQ ID NO: 1 and having an activity of transferring glucose to fucose. The Preferably, it is a protein consisting of the amino acid sequence of amino acid residues 29 to 489 of SEQ ID NO: 1. In the present specification, the term “protein of the present invention” is not limited to the protein having the full length of the amino acid sequence shown in SEQ ID NO: 1, but includes a protein containing a partial sequence of the amino acid sequence.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments will be described in detail for the purpose of explaining the present invention.
ADVANTAGE OF THE INVENTION According to this invention, the transglycosylation agent containing the protein which has the activity which transfers glucose to a fucose by (beta) 1,3 bond, or the nucleic acid which codes it is provided.

(1) Glc-T protein As described above, our group succeeded in cloning a gene encoding an enzyme that transfers galactose to N-acetylglucosamine, and determined its base sequence and deduced amino acid sequence. (Japanese Patent Laid-Open No. 2004-215619). Thereafter, the present inventors added a protein having the same amino acid sequence or a partial sequence thereof as the galactose transferase (corresponding to “Gal-T7” disclosed in JP-A-2004-215619) in addition to the above activity. The present invention has been completed by finding that it has a stronger activity of transferring glucose to fucose by β1,3 bond.

  The Glc-T protein used for the glycosyltransferase of the present invention is typically represented by SEQ ID NO: 1 (corresponding to SEQ ID NO: 1 of JP-A-2004-215619) in the present specification. The base sequence of the nucleic acid that has an amino acid sequence and encodes it is shown in SEQ ID NO: 2 (corresponding to SEQ ID NO: 2 of JP-A 2004-215619).

  In one embodiment of the present invention, the Glc-T protein used in the glycosyltransferase of the present invention has one or more amino acid substitutions or deletions in SEQ ID NO: 1, or one or more in the sequence. It may be a protein having an amino acid sequence into which is added or added. Also, such an amino acid sequence is at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90% of the amino acid sequence shown in SEQ ID NO: 1. Have identity.

  According to the present invention, a protein having an amino acid sequence in which all or part of amino acid residues 1-28 is deleted from the amino acid sequence of SEQ ID NO: 1 is provided. Preferably, it is a protein consisting of the amino acid sequence of amino acid residues 29 to 498 in SEQ ID NO: 1. In the present specification, a protein having these partial sequences may be referred to as a “short-chain Glc-T protein”. These short-chain Glc-T proteins can be used for the glycosyl transfer agent of the present invention as long as they have an activity of transferring glucose to fucose through β1,3 bonds.

  The Glc-T of the present invention has a cluster of hydrophobic amino acid residues on the N-terminal side of the amino acid sequence, and a transmembrane region or a signal sequence may be considered. The region of amino acid residues 29 to 498 excluding that region is predicted to be a region of glycosyltransferase activity. In this amino acid residue 29-498 region, there are three amino acid motifs (beta3GT motif) conserved in the glycosyltransferase family that transfers sugar by β3 bond, and the sequence between the motifs is β1,3N-acetylglucosamine 27%, 26%, and 23% homology with m-Fringe, which is a transferase, core1 Gal-T1, which is a β1,3-galactosyltransferase, and ChSy, which is a β1,3-glucuronyltransferase, respectively. In the second motif in the beta3GT motif, there is a DDD that seems to correspond to a DXD sequence required for binding to a divalent cation.

  In one embodiment of the present invention, the short-chain Glc-T protein used for the glycosyl transfer agent of the present invention has one or more amino acids in the amino acid sequence of amino acid residues 29 to 498 in SEQ ID NO: 1. It may be a protein that has an amino acid sequence substituted or deleted, or having one or more amino acids inserted or added in the sequence. Also, such an amino acid sequence is at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90% of the amino acid sequence shown in SEQ ID NO: 1. Have identity.

In the present specification, for the understanding of the present invention, the properties of Glc-T and short-chain Glc-T used in the glycosyltransferase of the present invention will be described below.
Action : Transfers glucose to fucose at the non-reducing end by β1,3 bond. When the reaction to be catalyzed is described by the reaction formula,
Fucose-R + UDP-Glucose → Glucose-Fucose-R + UDP
(Fuc-R + UDP-Glc → Glc-Fuc-R + UDP)
Substrate specificity : fucose, for example fucose-R (R is serine, threonine, sugar chain, peptide, protein, benzyl group, paranitrophenyl group, orthonitrophenyl group, orthomethoxyphenyl group).

  According to the present invention, a protein having an activity of transferring glucose to fucose is provided. As long as the protein of this invention has the characteristics described in this specification, the origin, manufacturing method, etc. are not limited. That is, the protein of the present invention may be a naturally occurring protein, a protein expressed from recombinant DNA by genetic engineering techniques, or a chemically synthesized protein.

  The protein of the present invention typically has an amino acid sequence consisting of 498 amino acid residues set forth in SEQ ID NO: 1. However, among natural proteins, there are 1 to more than one due to differences in the varieties of the species that produce them, gene mutations due to differences in ecotypes, or the presence of similar isozymes. It is well known that mutant proteins with single amino acid mutations exist. As used herein, the term “mutant protein” means that one or more amino acids are substituted or deleted in the amino acid sequence shown in SEQ ID NO: 1, or one or more amino acids are added to the amino acid sequence. It means a protein having an amino acid sequence in which an amino acid is inserted or added and having an activity of transferring glucose to fucose. Here, the “plurality” is preferably 1-100, more preferably 1-50, and most preferably 1-20. In general, when amino acids are substituted by site-specific mutation, the number of amino acids that can be substituted to such an extent that the activity of the original protein is conserved is preferably 1-10.

  The protein of the present invention has the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 (bottom) based on the estimation from the base sequence of the cloned nucleic acid, but is not limited to only the protein having the sequence. Rather, it is intended to include all homologous proteins as long as they have the characteristics described herein. The identity is at least 30% or more, preferably 50% or more, more preferably 70% or more, further preferably 80% or more, and most preferably 90% or more.

  In the present specification, the percent identity is determined by, for example, the BLAST program described in Altschul et al. (Nucl. Acids. Res. 25., p. 3389-3402, 1997) or Pearson et al. (Proc. Natl. Acad). Sci. USA, p. 2444-2448, 1988) can be determined by comparison with sequence information. The program can be used on the Internet from the website of National Center for Biotechnology Information (NCBI) or DNA Data Bank of Japan (DDBJ). Various conditions (parameters) for identity search by each program are described in detail on the site, and some settings can be changed as appropriate, but the search is usually performed using default values. It should be noted that other programs for sequence comparison used by those skilled in the art can also be used.

  In general, substitution between amino acids having similar properties (for example, substitution from one hydrophobic amino acid to another hydrophobic amino acid, substitution from one hydrophilic amino acid to another hydrophilic amino acid, substitution from one acidic amino acid to another When a substitution with an acidic amino acid or a substitution from one basic amino acid to another basic amino acid is introduced, the resulting mutant protein often has the same properties as the original protein. Techniques for producing recombinant proteins having such desired mutations using gene recombination techniques are well known to those skilled in the art, and such mutant proteins are also included within the scope of the present invention.

  The protein having the amino acid sequence represented by SEQ ID NO: 1 of the present invention can be obtained according to the method disclosed in JP-A-2004-215619. Moreover, those skilled in the art can easily prepare the protein of the present invention having an amino acid sequence from which all or part of amino acid residues 1-28 of SEQ ID NO: 1 has been deleted according to a conventional method. The protein of the present invention having an amino acid sequence from which amino acid residues 1-28 of SEQ ID NO: 1 have been deleted is, for example, according to Example 1 described below, as shown in SEQ ID NO: 2 showing a nucleic acid encoding the protein of the present invention. A large amount of the protein can be obtained by introducing and expressing the base sequence into Escherichia coli, yeast, insect, or animal cells using an expression vector that can be amplified in each host.

  According to the present invention, the amino acid sequence of these proteins and the DNA sequence encoding the protein or a part of the protein are used in the same manner from other species using genetic engineering techniques such as nucleic acid amplification reactions such as hybridization and PCR. It is possible to easily isolate a gene encoding a protein having the above physiological activity. In such a case, novel proteins encoded by these genes are also included in the scope of the present invention.

The protein of the present invention has the amino acid sequence as described above, and a sugar chain may be bound to the protein as long as it has the enzyme activity.
As described in Example 2 below, the protein of the present invention has an activity of transferring Glc to Fuc from the search for an acceptor substrate and a donor substrate in the protein of the present invention. More specifically, the protein of the present invention has the following properties:

(A) Specificity of acceptor substrate The acceptor substrate is not particularly limited as long as the protein of the present invention is a substrate capable of transferring a glucose residue from a glucose donor substrate. Preferably, Fucα-R, Fucα1-2Galβ1 -4GlcNAcβ-R (hereinafter sometimes referred to as “H antigen type 2”), Galβ1-3 (Fucα1-4) GlcNAcβ-R (hereinafter referred to as “Le ^a ”), Fucα1-2Galβ1-3GlcNAcβ-R (hereinafter referred to as “H antigen type 2”) “H antigen type 1”), Galβ1-4 (Fucα1-3) GlcNAcβ-R (hereinafter, “Le ^x ”), Fucα1-2Galβ1-3 (Fucα1-4) GlcNAcβ-R (hereinafter, “Le ^b ”), GalNAcα1-3 (Fucα1-2) Galβ1-3GlcNAcβ-R (hereinafter “A antigen”), Galα1-3 ( ucα1-2) Galβ1-3GlcNAcβ-R (hereinafter, "B antigen"), more preferably Fucα-R, H antigen type 2, Le ^a, even more preferably Fucα-R. Abbreviations used here are Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine. R is not limited, but is typically serine, threonine, sugar chain, peptide, protein, benzyl (Bz) group, paranitrophenyl (pNp) group, orthonitrophenyl group, or orthomethoxyphenyl group. .

(B) Specificity of donor substrate The donor substrate is preferably glucose as a substrate that can be transferred to the aforementioned acceptor substrate by the protein of the present invention. More preferably, it is a sugar nucleotide having glucose. Sugar nucleotides include uridine diphosphate-glucose (UDP-Glc), adenosine diphosphate-glucose (ADP-Glc), guanosine diphosphate-glucose (GDP-Glc), cytidine diphosphate-glucose (CDP-). Glc) and the like are exemplified, and UDP-Glc is most preferable.

(C) Tissue expression The protein of the present invention is expressed in mammalian tissue, preferably human tissue.
(I) Preferably, high expression is shown in the testis and uterus.
(Ii) Preferably shows moderate expression in the brain, spinal cord, fetal brain, adrenal gland, thyroid, spleen, skeletal muscle, lung, stomach, large intestine, pancreas.
(Iii) Preferably low expression in cerebellum, bone marrow, thymus, heart, trachea, mammary gland, liver, fetal liver, salivary gland, small intestine, kidney, prostate, placenta.

(2) Isolation / purification of Glc-T protein The protein of the present invention having the full length of the amino acid sequence shown in SEQ ID NO: 1 is prepared according to the Gal-T7 protein preparation method disclosed in JP-A-2004-215619. It can be expressed and isolated / purified.

  A protein having an amino acid sequence in which all or part of amino acid residues 1-28 is deleted from the amino acid sequence shown in SEQ ID NO: 1 is described in this specification and specially described as described in Example 1 below. Since a base sequence encoding a protein consisting of the full-length amino acid sequence of the present invention is disclosed in Japanese Unexamined Patent Application Publication No. 2004-215619, those skilled in the art can incorporate a part of the base sequence into a predetermined expression vector, Is introduced into a host cell, and the host cell is cultured under appropriately selected culture conditions, so that the target protein can be expressed in large quantities.

  The protein of the present invention can be obtained from a culture obtained by culturing the above host cells based on the following method. That is, when the protein of the present invention accumulates in a host cell, the host cell is collected by an operation such as centrifugation or filtration, and this is collected with an appropriate buffer (for example, a Tris buffer having a concentration of about 10 mM to 100 mM, phosphorous). Acid buffer, HEPES buffer, MES buffer, etc. A method suitable for the host cell to be used after suspending in a buffer whose pH varies depending on the buffer used, but preferably in the range of pH 5.0 to 9.0. The cells are disrupted by centrifugation and the contents of the host cells are obtained by centrifugation. On the other hand, when the protein of the present invention is secreted outside the host cell, the host cell and the medium are separated by an operation such as centrifugation or filtration to obtain a culture filtrate. The host cell disruption solution or culture filtrate can be used for isolation or purification of the protein of the present invention as it is or after performing ammonium sulfate precipitation and dialysis. Examples of the isolation / purification method include the following methods. That is, when a tag such as 6 × histidine, GST, or maltose binding protein is attached to the protein, a method by affinity chromatography suitable for each tag generally used can be mentioned. On the other hand, when the protein of the present invention is produced without attaching such a tag, a method by ion exchange chromatography can be mentioned. In addition to this, a method of combining gel filtration, hydrophobic chromatography, isoelectric point chromatography and the like can also be mentioned.

  The proteins of the present invention may also include peptides added to facilitate purification and identification. Such peptides include, for example, the antigenic identification peptides described in Poly-His or US Pat. No. 5,011,912 and Hopp et al., Bio / Technology, 6: 1204, 1988. One such peptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 3), which is highly antigenic and specific The monoclonal antibody provides an epitope to which it binds reversibly, allowing rapid assay and easy purification of the expressed recombinant protein. The murine hybridoma designated 4E11 is a FLAG® peptide in the presence of certain divalent metal cations, as described in US Pat. No. 5,011,912, incorporated herein by reference. Monoclonal antibodies that bind to The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under the deposit number HB 9259. Monoclonal antibodies that bind to the FLAG® peptide are available from Eastman Kodak Co. , Scientific Imaging Systems Division, New Haven, Connecticut.

  Specifically, as described in Example 1 to be described later, a FLAG cDNA is inserted into an expression vector that expresses the protein of the present invention, and a FLAG-labeled protein is expressed. Can be confirmed.

(3) Antibodies that Bind to Glc-T Protein Provided herein are antibodies that are immunoreactive with the proteins of the present invention. Such an antibody binds to the protein via the antigen binding site of the antibody. Therefore, the protein, fragment, mutant, fusion protein and the like of SEQ ID NO: 1 as described above can be used as an “immunogen” in producing an antibody that is immunoreactive with it. More specifically, proteins, fragments, variants, fusion proteins, etc. contain antigenic determinants or epitopes that elicit antibody formation. These antigenic determinants or epitopes can be either linear or conformational (intermittent). The antigenic determinant or epitope may be identified by any method known in the art.

  Accordingly, one aspect of the present invention relates to antigenic epitopes of the proteins of the present invention. Such epitopes are useful for making antibodies, particularly monoclonal antibodies, as described in more detail below. Furthermore, the protein-derived epitopes of the invention can be used in assays and as research reagents to purify antibodies that specifically bind from substances such as polyclonal antibodies (antisera) or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques known in the art, such as solid phase synthesis, chemical or enzymatic cleavage of proteins, or using recombinant DNA techniques. .

  For antibodies that may be induced by the proteins of the invention, both polyclonal and monoclonal antibodies, whether all or part of the protein or epitopes are isolated, It can be prepared by conventional techniques. See, for example, Kennet et al. (Supervised), Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, New York, 1980.

  Hybridoma cell lines that produce monoclonal antibodies specific for the proteins of the invention are also contemplated herein. Such hybridomas can be produced and identified by conventional techniques. One method for producing such a hybridoma cell line is to immunize an animal with the protein; to collect spleen cells from the immunized animal; to fuse the spleen cells to a myeloma cell line, thereby And identifying a hybridoma cell line that produces a monoclonal antibody that binds to the protein. Monoclonal antibodies can be recovered by conventional techniques.

  Monoclonal antibodies of the present invention include chimeric antibodies, eg, humanized forms of murine monoclonal antibodies. Such humanized antibodies may be prepared by known techniques and may provide the benefit of reduced immunogenicity when the antibody is administered to a human. In one embodiment, the humanized monoclonal antibody comprises a murine antibody variable region (or only its antigen binding site) and a constant region from a human antibody. Alternatively, the humanized antibody fragment may comprise an antigen binding site of a murine monoclonal antibody and a variable region fragment derived from a human antibody (which lacks an antigen binding site).

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. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.

  In one embodiment, the antibody is specific for a protein of the invention and does not cross-react with other proteins. Screening methods capable of identifying such antibodies are known and may involve, for example, immunoaffinity chromatography.

  The antibodies of the invention can be used in assays that detect the presence of the proteins or fragments of the invention, either in vitro or in vivo. The antibodies can also be used in purifying the polypeptides or fragments of the invention by immunoaffinity chromatography.

  In addition, antibodies that can block the binding of a protein of the invention to a binding partner, such as a receptor substrate, can be used to inhibit biological activity resulting from such binding. Such blocking antibodies may be identified using any suitable assay, such as by testing the antibody for its ability to inhibit binding of the protein to specific cells expressing the receptor substrate. . Alternatively, blocking antibodies can be identified in an assay for the ability to inhibit a biological effect resulting from a protein of the invention that is bound to a binding partner of a target cell.

  Such antibodies can be used in vitro or administered in vivo to inhibit biological activity mediated by the entity that produced the antibody. Thus, it is possible to treat disorders caused or exacerbated (directly or indirectly) by the interaction of a protein of the invention with a binding partner. The therapy involves administering to a mammal in vivo an amount of a blocking antibody effective to inhibit binding partner-mediated biological activity. In general, monoclonal antibodies are preferred for use in such therapies. In one embodiment, antigen binding antibody fragments are used.

(4) Nucleic acid encoding Glc-T protein According to the present invention, there is provided a glycosyl transfer agent that transfers a glucose to fucose by β1,3 bond, including a nucleic acid encoding Glc-T protein. The nucleic acid also includes both single-stranded and double-stranded DNA, and its RNA complement. DNA includes, for example, naturally-derived DNA, recombinant DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. As the nucleic acid to be used, DNA is preferable. In this specification, when describing a specific base sequence, its complementary strand is also included unless otherwise specified.

  The nucleic acid used for the sugar transfer agent of the present invention is preferably a nucleic acid (including its complement) encoding the human amino acid sequence represented by SEQ ID NO: 1. Typically, it has the base sequence of SEQ ID NO: 2 (including its complement). A person skilled in the art knows that there are a small number of mutations in natural nucleic acids due to differences in varieties of species that produce them, differences in ecotypes, and the presence of similar isozymes. Is well known. The nucleic acid that can be used is not limited to the nucleic acid having the base sequence shown in SEQ ID NO: 2, but includes all nucleic acids that encode Glc-T protein. The nucleic acid used for the sugar transfer agent of the present invention preferably has nucleotides 1-1494, more preferably nucleotides 85-1494 in the base sequence of SEQ ID NO: 2.

As used herein, “under stringent conditions” means to hybridize under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by those skilled in the art having general techniques based on, for example, the length of the DNA. Basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Chapter 6-7, Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, 5 × SSC, 0.5 Pre-wash solution of% SDS, 1.0 mM EDTA, pH 8.0, about 50% formamide at about 40-50 ° C., 2 × SSC-6 × SSC (or about 50% formamide at about 42 ° C. , Other similar hybridization solutions such as Stark's solution), and the use of washing conditions of about 60 ° C., 0.5 × SSC, 0.1% SDS. Preferably moderately stringent conditions include hybridization conditions of about 50 ° C. and 2 × SSC. High stringency conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA. In general, these conditions include hybridization at higher temperatures and / or lower salt concentrations than moderately stringent conditions (eg, about 65 ° C., 6 × SSC to 0.2 × SSC, preferably 6 × SSC, More preferably 2 × SSC, most preferably 0.2 × SSC hybridization) and / or washing, eg hybridization conditions as described above, and approximately 68 ° C., 0.2 × SSC, 0.1% Defined with SDS washing. In hybridization and wash buffers, SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) to SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH ₂ PO ₄ , and 1. 25 mM EDTA, pH 7.4) can be substituted, and washing is performed for 15 minutes after hybridization is complete. In order to achieve the desired degree of stringency by applying the basic principles known to those skilled in the art and governing the hybridization reaction and duplex stability, as further described below, the wash temperature It should be understood that the wash salt concentration can be adjusted as needed (see, eg, Sambrook et al., 2001). When hybridizing a nucleic acid to a target nucleic acid of unknown sequence, the hybrid length is assumed to be that of the hybridizing nucleic acid. When hybridizing nucleic acids of known sequence, the length of the hybrid can be determined by juxtaposing the nucleic acid sequences and identifying the region or regions with optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be the length of less than 50 base pairs, not should be 5-25 less ℃ than hybrid melting temperature (T _m), T m is determined by the following equation Is done. For hybrids less than 18 base pairs in length, T _m (° C.) = 2 (A + T base number) +4 (G + C base number). For hybrids longer than 18 base pairs, T _m = 81.5 ° C. + 16.6 (log ₁₀ [Na ⁺ ]) + 41 (molar fraction [G + C]) − 0.63 (% formamide) −500 / n, where N is the number of bases in the hybrid, and [Na ⁺ ] is the sodium ion concentration in the hybridization buffer (1 × SSC [Na ⁺ ] = 0.165 M). Preferably, each such hybridizing nucleic acid is at least 8 nucleotides (or more preferably at least 15 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides), or at least 1% of the length of the nucleic acid to which it hybridizes (more preferably at least 25%, or at least 50%, or at least 70%, and most preferably at least 80) At least 50% (more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least with the nucleic acid to which it hybridizes 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%). Here, sequence identity, as described in more detail above, by comparing the sequences of hybridizing nucleic acids aligned in parallel to maximize identity with overlapping portions while minimizing sequence gaps. It is determined.

  Nucleic acid amplification reactions include, for example, polymerase chain reaction (PCR) (Saiki RK, et al., Science, 230, 1350-1354 (1985)), Ligates chain reaction (LCR) (Wu DY, et. al., Genomics, 4, 560-569 (1989); Barringer KJ, et al., Gene, 89, 117-122 (1990), Barany F., Proc. Natl. Acad. Sci. 189-193 (1991)) and transcription based amplification (Kwoh DY, et al., Proc. Natl. Acad. Sci. USA, 86, 1173-1177 (1989)) Reaction, as well as strand displacement reaction (SDA) (Walker GT et al., Proc. Natl. Acad. Sci. USA, 89, 392-396 (1992); Walker GT, et al., Nuc. Acids. Res., 20, 1691-1696 (1992)), Self-sustained sequence replication (3SR) (Guatelli JC, Proc. Natl. Acad. Sci. USA, 87, 1874-1878 (1990)) and Qβ replicase system (Lizardi et al., BioTechnology 6, p.1197-1202 ( 1988)) and the like. Further, amplification based on a nucleic acid sequence by competitive amplification of a target nucleic acid and a mutant sequence described in European Patent No. 0525882 (Nucleic Acid Sequence Based Amplification: NASBA) reaction or the like can also be used. The PCR method is preferred.

  The homologous nucleic acid cloned using the above hybridization, nucleic acid amplification reaction or the like is at least the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing or the nucleotide sequence of nucleotides 85-1494 in SEQ ID NO: 2. It has an identity of 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90%.

  The percent nucleic acid identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, this comparison is made by comparing sequence information using a computer program. A typical preferred computer program is the Wisconsin package, version 10.0 program “GAP” from the Genetics Computer Group (GCG; Madison, Wis.) (Devereux et al., 1984, Nucl. Acids Res. 12: 387). By using this “GAP” program, in addition to comparing two nucleic acid sequences, two amino acid sequences can be compared, and a nucleic acid sequence and an amino acid sequence can be compared. Here, the preferred default parameters of the “GAP” program are: (1) GCG implementation of a unitary comparison matrix for nucleotides (including values of 1 for identical and 0 for non-identical), and Schwartz and Dayhoff supervised “Atlas of Polypeptide Sequence and Structure” National Biomedical Research Foundation, pages 353-358, 1979, Gribskov and Burgess, Nucl. Acids Res. 14: 6745, 1986 weighted amino acid comparison matrix; or other comparable comparison matrix; (2) 30 penalties for each gap of amino acids and one additional penalty for each symbol in each gap; or each of the nucleotide sequences Includes 50 penalties for gaps and an additional 3 penalties for each symbol in each gap; (3) no penalty to end gaps; and (4) no maximum penalty for long gaps. Other sequence comparison programs used by those skilled in the art include, for example, the National Medical Library website: http: // www. ncbi. nlm. nih. gov / blast / bl2seq / bls. The BLASTN program available for use by html, version 2.2.7, or the UW-BLAST 2.0 algorithm can be used. Standard default parameter settings for UW-BLAST 2.0 can be found at the following Internet site: http: // blast. Wustl. It is described in edu. In addition, the BLAST algorithm uses a BLOSUM62 amino acid scoring matrix and the selection parameters that can be used are: (A) Segments of query sequences with low compositional complexity (Woughton and Federhen SEG program (Computers and Chemistry, 1993); Woton and Federhen, 1996, “Analysis of compositionally biased regions in sequence data bases” or 71: 66. , Segments consisting of short-periodic internal repeats (Cl including a filter for masking verie and States (determined by the XNU program of Computers and Chemistry, 1993), and (B) a threshold of statistical significance for reporting a fit to the database sequence, or According to the statistical model of E-score (Karlin and Altschul, 1990), the expected probability of a match that is simply found by chance; if the statistical significance due to a match is greater than the E-score threshold, this fit is Not reported); the preferred E-score threshold value is 0.5 or, in order of increasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e 30,1e-40,1e-50,1e-75, or a 1e-100.

(5) Nucleic acid for measuring nucleic acid encoding Glc-T protein According to the present invention, a nucleic acid that hybridizes with a nucleic acid encoding the Glc-T protein of the present invention (hereinafter referred to as "measuring nucleic acid") Is provided. The measurement nucleic acid of the present invention is typically a naturally-derived or synthesized fragment of a nucleic acid encoding the Glc-T protein of the present invention, and includes, but is not limited to, a primer or a probe. Absent. Note that the term “measurement” used in the present specification includes any of assay, detection, amplification, quantification, and semi-quantification.

(A) Primer When the measurement nucleic acid of the present invention is used as a primer for nucleic acid amplification reaction, the measurement nucleic acid of the present invention is an oligonucleotide,
Based on SEQ ID NO: 2 showing the base sequence of the gene encoding Glc-T protein, two regions are selected so as to satisfy the following conditions:
1) The length of each region is 15-50 bases;
2) The proportion of G + C in each region is 40-70%;
A single-stranded DNA having the same base sequence as the above region or a base sequence complementary to the above region is produced, or the genetic code is degenerate so as not to change the amino acid residue encoded by the single-stranded DNA. By producing a mixture of the considered single-stranded DNA and, if necessary, producing the single-stranded DNA modified so as not to lose the binding specificity to the base sequence of the gene encoding the protein. The manufactured oligonucleotide is provided.

The primer of the present invention preferably has a sequence homologous to the partial region of the nucleic acid of the present invention, but there may be a mismatch of 1 or 2 bases.
The number of bases of the primer of the present invention is 15 bases or more, preferably 18 bases or more, more preferably 21 bases or more, and 50 bases or less.

  The primer of the present invention typically has the nucleotide sequence of SEQ ID NO: 4-6, and can be used alone or as a primer pair in which two appropriate types are combined. This nucleotide sequence is designed as a primer for PCR for cloning a gene fragment encoding each protein based on the amino acid sequence of SEQ ID NO: 1, and all the bases capable of encoding the amino acid. Is a mixed primer.

(B) Probe When the nucleic acid for measurement of the present invention is used as a probe, the nucleic acid for measurement of the present invention preferably has a sequence homologous to the whole or partial region of the base sequence described in SEQ ID NO: 2. When the nucleic acid for measurement of the present invention is a cDNA probe, the number of bases is 15 bases or more, preferably 20 bases or more, and the longest is the entire length of the coding region, that is, 1497 bases. Even if there is a mismatch of 20% or less, preferably 10% or less, with the base sequence described in SEQ ID NO: 2 or its complementary base sequence, it can function as a probe. When the nucleic acid for measurement of the present invention is a synthetic oligonucleotide, the number of bases is 15 bases or more, preferably 20 bases or more, more preferably 24 bases or more. In the case of a synthetic oligonucleotide, depending on the length, even if there is a mismatch of about 1 or 2 bases with the base sequence shown in SEQ ID NO: 2 or its complementary base sequence, it can function as a probe.

  The probe of the present invention has the base sequence described in SEQ ID NO: 7, for example. This corresponds to base numbers 1328 to 1351 of SEQ ID NO: 2. The probe may be obtained from a naturally-occurring nucleic acid by restriction enzyme treatment, or may be a synthesized oligonucleotide.

  The probe of the present invention includes a labeled probe with a label such as a fluorescent label, a radiolabel, or a biotin label in order to detect or confirm that the probe has hybridized with the target sequence. It is possible to determine whether the test nucleic acid is present in the sample by immobilizing the test nucleic acid or its amplified product, hybridizing with the labeled probe, washing, and measuring the label bound to the solid phase. it can. Alternatively, it is possible to immobilize the measurement nucleic acid, hybridize the test nucleic acid, and detect the test nucleic acid bound to the solid phase with a labeled probe or the like. In such a case, the measurement nucleic acid bound to the solid phase is also called a probe.

  In general, nucleic acid amplification methods such as PCR are well known in this field, and reagent kits and devices therefor are also commercially available and can be easily performed. When the nucleic acid amplification method is performed using the above-described pair of measurement nucleic acids of the present invention as a primer and the test nucleic acid as a template, the test nucleic acid is amplified, whereas the test nucleic acid is contained in the sample. In the absence of amplification, amplification does not occur, and it can be determined whether or not the test nucleic acid is present in the sample by detecting the amplification product. For amplification product detection, the reaction solution after amplification is electrophoresed, and the band is stained with ethidium bromide, etc., or the amplification product after electrophoresis is immobilized on a solid phase such as nylon membrane, so that it is specific to the test nucleic acid. This can be carried out by hybridizing with a labeled probe that hybridizes to, and detecting the label after washing. It is also possible to quantify the amount of test nucleic acid in a sample by performing so-called real-time detection PCR using a quencher fluorescent dye and a reporter fluorescent dye. In addition, since the kit for real-time detection PCR is also marketed, it can carry out easily. Furthermore, the test nucleic acid can be semi-quantified based on the intensity of the electrophoresis band. The test nucleic acid may be mRNA or cDNA reverse transcribed from mRNA. When amplifying mRNA as a test nucleic acid, the NASBA method (3SR method, TMA method) using the above-mentioned pair of primers can also be employed. The NASBA method itself is well known, and kits therefor are also commercially available. Therefore, the NASBA method can be easily carried out using the above pair of primers.

(6) Glycosyltransferase Agent According to the present invention, a glycosyltransferase agent comprising the protein (glucose transferase) defined above or a nucleic acid encoding the same is provided. As the glycosyltransferase of the present invention, glucose transferase (Glc-T) may be used directly, or a nucleic acid encoding Glc-T inserted into an appropriate expression vector, for example. Good.

(7) Knockout animal Since it became clear that the expression of Glc-T is involved in the expression of cancer according to the present invention, the expression of the gene encoding Glc-T protein is partially or completely suppressed in experimental animals. By doing so, it is also possible to prepare knockout animals used for elucidation of detailed cancer metastasis mechanisms in vivo. As the knockout animal, for example, mammals such as mice, rats, rabbits, dogs, cats, monkeys and the like are preferable, and mice or rats are most preferable because methods for preparing knockout animals are established to some extent. Not done. For example, knockout mice can be performed according to the description of “the latest technology of gene targeting” (edited by Ken Yagi, Yodosha, 2000), gene targeting (translated by Tetsuo Noda, Medical Science International, 1995), etc. it can. A knockout mouse can also be produced by a “method for suppressing gene expression” such as the small interfering RNA method (Brummelkamp, TR et al., Science, 296, 5501-553 (2002)).

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1 Preparation of Glc-T Preparation of short chain type Glc-T (1) Incorporation of short chain type Glc-T gene into expression vector In order to create an expression system of short chain type Glc-T, first, the Glc-T gene was transferred to Sigma pFLAG-CMV1. PFALG-Glc-T was prepared by integrating into a vector. Two types of primers GP-1052 (5′-ggaattctgaagatagaagaagaagagt-3 ′) (SEQ ID NO: 4) (SEQ ID NO: 4) (nucleotide 5-28 corresponds to nucleotides 84-107 of SEQ ID NO: 2) designed according to the base sequence shown in SEQ ID NO: 2 ) And GP-1049 (5′-gctctagagtgtattataactcctctcgaaaa-3 ′) (SEQ ID NO: 5) (nucleotide 14-32 corresponds to nucleotide 1497-1479 of SEQ ID NO: 2), and a DNA fragment necessary for enzyme activity is Amplification was performed using Marathon-Ready cDNA (Human Brain) as a template. Since these primers contain a restriction enzyme Eco RI or Xba I cleavage site, it is possible to insert a PCR amplified fragment into the EcoRI-Xba I site of the pFLAG-CMV1 vector. However, since there is one Eco RI in the PCR-amplified fragment, first, the 3 ′ Eco RI-Xba I fragment and then the 5 ′ Eco RI-Eco RI fragment are inserted by a conventional method. -A T expression vector was constructed. The produced vector was named pFLAG-Glc-T. By this operation, Glc-T continues to the FLAG tag, and protein is produced from the 29th serine.

(2) Transfection and Recombinant Enzyme Expression 2 × 10 ⁶ HEK293 cells were cultured overnight in DMEM (Dulbecco's modified Eagle's medium) containing 10% FCS (fetal bovine serum) in advance, and then Lipofectamine 2000 (Invitrogen) 15 μg of pFLAG-Glc-T was introduced into the cells according to the company's protocol. The culture supernatant after 48 to 72 hours of culture was collected, and NaN ₃ (0.05%), NaCl (150 mM), CaCl ₂ (2 mM), anti-M1 resin (Sigma) (50 μl) was added to 10 ml of the supernatant. Mix and stir at 4 ° C. overnight. The reaction mixture is centrifuged (3000 rpm, 5 minutes, 4 ° C.) to collect the pellet, 900 μl of ₂ mM CaCl ₂ · TBS is added and centrifuged again (2000 rpm, 5 minutes, 4 ° C.). The sample was suspended in 1 mM CaCl ₂ · TBS to obtain a sample for activity measurement (Glc-T enzyme solution).
The enzyme solution was subjected to SDS-PAGE and Western blotting by a conventional method to confirm that the target protein was expressed. Antibody is anti-FLAG
M2-peroxidase (A-8592, SIGMA) was used.

2. Preparation of full length Glc-T (1) Incorporation of full length Glc-T gene into expression vector Full length Glc-T is composed of two kinds of primers GP-1053 (5'-ggaattcaggatgcggccgcccgcctgct-3 ') (SEQ ID NO: 6) (nucleotide 11- 29 corresponds to nucleotides 1-19 of SEQ ID NO: 2) and GP-1049, and the full-length DNA fragment was amplified using Marathon-Ready cDNA (Human Brain) as a template. These primers contained a restriction enzyme Eco RI or Xba I cleavage site, and were ligated to pcDNA3.1 (+) of Invitrogen by the same method as described above to construct pcDNA3.1-Glc-T.

(2) Transfection and expression of recombinant enzyme 2 × 10 ⁶ CHO cells were pre-cultured overnight in RPMI 1640 previously containing 10% FCS (fetal calf serum) and then Lipofectamine 2000 (Invitrogen) according to the protocol of the company. Into the cells, 15 μg of pcDNA3.1-Glc-T was introduced. The next day, 0.8 mg / ml G418 was added to the medium, and the transfectants were selected by further culturing for several days. 1 × 10 ⁶ Glc-T-introduced CHO cells were washed with PBS, suspended in TBS Buffer containing 1% Nonidet P40, Proteinase Inhibitor Cocktail (Sigma), and sonicated at 4 ° C. for 15 minutes. This was centrifuged at 14,000 × G for 10 minutes at 4 ° C., and the supernatant was used as a cell extract, which was used as an enzyme source for measuring intracellular Glc-T activity.

Example 2 Substrate specificity of Glc-T protein Substrate specificity of short-chain Glc-T protein (1) Search for donor substrate Short-chain Glc-T using 5 μl of enzyme solution and various donor substrates for a mixture of various monosaccharide acceptor substrates A search for protein donor substrates was performed (Table 1). Acceptor substrates are Gal-α-pNp, Gal-β-oNp, GalNAc-α-Bz, GalNAc-β-pNp, GlcNAc-α-pNp, GlcNAc-β-Bz, Glc-α-pNp, Glc-β -PNp, GlcA-β-pNp, Fuc-α-pNp, Man-α-pNp (above, CALBIOCHEM), Xyl-α-pNp, Xyl-β-pNp (above, Sigma) each 2.5 nmol / Prepared to contain 20 μl. Here, “Gal” represents a D-galactose residue, “Glc” represents a D-glucose residue, “Fuc” represents a D-fucose residue, and “Man” represents D-mannose. “PNp” represents a p-nitrophenyl group, “Bz” represents a benzyl group, and “−” represents a glycosidic bond. “Α” and “β” are anomers of the glycosidic bond at the 1-position of the sugar ring, and “α” indicates that the positional relationship with the 5-position CH ₂ OH or CH ₃ is trans, and “cis” indicates “ β ”. Reaction solutions for various donor substrates (UDP-Glc, UDP-GalNAc, UDP-GlcNAc, UDP-Gal, GDP-Man, UDP-GlcA, UDP-Xyl and GDP-Fuc, all of which are Sigma) are shown in Table 1. It is as shown in.

An appropriate amount of ¹⁴ C-labeled donor substrate was also mixed in the reaction solution. All reaction times were 16 hours. After the reaction, the donor substrate having unreacted radioactivity was removed using a SepPack C18 column (Waters), and the radioactivity derived from the donor incorporated into the acceptor was measured with a liquid scintillation counter. As a result, Glc-T was able to detect significant activity only when UDP-Glc was used as a donor substrate (Table 2).

(2) Search for receptor substrate For the purpose of searching for a receptor substrate, each receptor substrate was used alone (10 nmol / 20 μl) and a reaction was performed. As a result, significant radioactivity was detected for Glc-T only when Fuc-α-pNp was used. From the above results, it was revealed that Glc-T is a glycosyltransferase that transfers Glc to Fuc (Table 3).

Furthermore, in order to examine the specificity of the receptor substrate containing Fuc, the activity of Glc-T was measured using the receptor substrate described in Table 4. The activity of transferring glucose to each substrate by Glc-T was evaluated relative to the case where Fucα-pNp was used as 100%. From the results of Table 4, Glc-T is not only Fucα-pNp but also at least Fucα1-2Galβ1-4GlcNAcβ-pNp (H antigen type 2) (activity 61%), Galβ1-3 (Fucα1-4) GlcNAcβ-pNp ( It was revealed that Le ^a ) (activity 52%) showed substrate specificity.

2. Substrate specificity of full-length Glc-T protein When a CHO cell extract containing an endogenous β3Glc-T activity is used as an enzyme source, as described above, Fuc-α-pNp and UDP-Glc It has been reported that Glcβ1,3Fuc-α-pNp is produced upon reaction (Moloney, DJ et al., Supra). Therefore, when the reaction product of the recombinant full length Glc-T of the present invention is developed by thin layer chromatography using chloroform: methanol: CaCl ₂ (65: 35: 8) as a developing solvent, the reaction product of the CHO cell extract is produced. Unfolded at the same position as the object. In addition, each reaction product is Trichoderma sp. Exo-1,3-β-glucanase and Almond sp. It was digested with β-glucosidase from (data not shown). Further, when CHO cells were transfected with full-length Glc-T recombined with pcDNA3.1 (+) (Invitrogen) and the transfectant cell extract was used as an enzyme source, the same reaction product as the CHO cell extract was obtained. The position radioactivity was increased (FIG. 1). From the above, it was suggested that the reaction product of full-length Glc-T of the present invention was Glcβ1,3Fuc-α-pNp, similar to the reaction product of the CHO cell extract.

Example 3 Analysis of Glc-T Expression in Human Tissue Using the cDNA of human normal tissue, the expression level of the gene was quantified by quantitative PCR. The normal tissue cDNA was reverse transcribed from Clontech total RNA. For cell lines, total RNA was extracted, and cDNA was prepared by a conventional method. The primers used for the quantitative expression analysis of the Glc-T gene were GP-K14-F2 (5′-gacacacaccccctctttcca-3 ′) (SEQ ID NO: 7) (corresponding to nucleotides 1305-1325 of SEQ ID NO: 2), GP-K14 -R2 (5'-tgggaacttgagagaagaggtagtc-3 ') (SEQ ID NO: 8) (corresponding to nucleotides 1378-1354 of SEQ ID NO: 2), the probe is GP-K14-P2 (5'-aggctcggccgggtgattacccta-3') (SEQ ID NO: 9 ) (Corresponding to nucleotides 1328-1351 of SEQ ID NO: 2). Universal PCR Master Mix was used for the enzyme and the reaction solution, and quantification was performed with an ABI PRISM 7700 Sequence Detection System (both Applied Biosystems) at a reaction solution volume of 25 μl. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a standard gene for quantification, and a standard curve for quantification was prepared with a template DNA at a known concentration to standardize the expression level of the gene. Moreover, pFLAG-Glc-T was used as the standard DNA for Glc-T. The reaction temperature was 50 ° C. for 2 minutes, 95 ° C. for 10 minutes and then 50 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute. The results are shown in FIG. Glc-T was highly expressed in the testis and uterus, but it could be confirmed in all tissues examined.

Example 4 Production of Knockout Mouse A target vector for the production of a knockout mouse was a 2 kb region containing the 11th exon containing the DDD sequence predicted to be essential for enzyme activity and the 10th exon adjacent thereto by PCR. Amplified and incorporated between two lox sequences as in FIG. By crossing with cre transgenic mice, this region is deleted in the knockout mice. For homologous recombination, the 3.7 kb and 3 kb regions on both sides were also amplified by PCR to construct a vector as shown in FIG. This vector also contains the DT3, Neo gene for selection of transfectants. This vector can be transfected into C57BL / 6-derived ES cells, and chimeric mice can be produced using the recombinant ES cells. The cre-lox system can be knocked out systemically, tissue-specific or cell-specifically.

  A glycosyl transfer agent containing Glc-T having a sufficient effect of transferring a glucose residue to a substrate having fucose at a non-reducing end is provided, whereby glucose can be efficiently applied to sugar chains, glycopeptides, glycoproteins, etc. Can be added.

FIG. 1 shows the analysis of the reaction product by Glc-T. The band shows the radioactive activity of [ ¹⁴ C] Glc incorporated into Fuc-α-pNp. The enzyme source in each lane was CHO cells transfected with lane 1: mock (negative control, lanes 2, 3: recombinant Glc-T, lanes 4, 5: CHO cell disruption extract, lanes 6, 7: Glc-T. The crushed extract was used, and lanes 3, 5 and 7 were subjected to exo-β1,3glucanase treatment on the reaction product. FIG. 2 is a graph showing the results of quantifying the expression level of Glc-T gene in various tissues by the real-time PCR method. The vertical axis represents the relative ratio of the Glc-T gene to the expression level of the control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. FIG. 3 shows a schematic diagram of a vector for generating a knockout mouse. A 2 kb region containing the 11th exon containing the DDD sequence expected to be essential for enzyme activity and the 10th exon adjacent to it were sandwiched by lox sequences. This region is knocked out by mating with a cre transgenic mouse in which cre is expressed in a tissue-specific or cell-specific manner.

Claims (11)

  A method of transferring glucose to fucose by β1,3 bond using a protein having an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1.   The amino acid sequence shown in SEQ ID NO: 1 or an amino acid in which 1 or 20 amino acids are substituted or deleted in the protein, or 1 or 20 amino acids are inserted or added to the amino acid sequence The method according to claim 1, which is a protein having a sequence.   The method of claim 1, wherein the protein has the amino acid sequence shown in SEQ ID NO: 1.   The method according to any one of claims 1 to 3, wherein all or part of amino acid residues 1-28 is deleted from the amino acid sequence of SEQ ID NO: 1.   The method according to claim 4, wherein the protein consists of an amino acid sequence of amino acid residues 29 to 498 in SEQ ID NO: 1. The protein has the following reaction:
Fucose-R + glucose-nucleotide → has activity of catalyzing glucose-fucose-R + nucleotide, where R is serine, threonine, sugar chain, peptide, protein, benzyl group, paranitrophenyl group, orthonitrophenyl 6. The method according to any one of claims 1 to 5, wherein the method is selected from the group consisting of a group and an orthomethoxyphenyl group. Possess an amino acid sequence having amino acid sequence homology of 90% or more of SEQ ID NO: 1, and, by using a protein having an activity of transferring at β1,3 bond glucose to fucose, glucose - fucose -R Where R is selected from the group consisting of serine, threonine, sugar chain, peptide, protein, benzyl group, paranitrophenyl group, orthonitrophenyl group, and orthomethoxyphenyl group. A method for synthesizing sugar chains.   A nucleic acid encoding a protein having an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1 is inserted into an expression vector, and a protein in which the protein encoded by the nucleic acid is expressed, fucose To transfer glucose to β1,3 bond.   The method according to claim 8, wherein the nucleic acid hybridizes with a nucleic acid having the base sequence of SEQ ID NO: 2 under stringent conditions in which hybridization is performed at 65 ° C. and 2 × SSC.   The method according to claim 8, wherein the nucleic acid has at least 95% sequence identity with a nucleic acid having the base sequence of SEQ ID NO: 2.   The method according to any one of claims 8 to 10, wherein the nucleic acid has nucleotides 85 to 1494 of the base sequence of SEQ ID NO: 2.