WO2000050608A1 - Nouveau polypeptide - Google Patents
Nouveau polypeptide Download PDFInfo
- Publication number
- WO2000050608A1 WO2000050608A1 PCT/JP2000/001070 JP0001070W WO0050608A1 WO 2000050608 A1 WO2000050608 A1 WO 2000050608A1 JP 0001070 W JP0001070 W JP 0001070W WO 0050608 A1 WO0050608 A1 WO 0050608A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polypeptide
- dna
- cells
- sugar chain
- gene
- Prior art date
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates to the synthesis of type 1 sugar chains such as sialyl Lewis a sugar chain in gastrointestinal cancer cells such as colon cancer cells and Teng carcinoma cells expressing sialyl Lewis a sugar chain.
- Novel polypeptide having galactosyltransferase activity method for producing the polypeptide, DNA encoding the polypeptide, recombinant vector into which the DNA has been incorporated, trait carrying the recombinant vector A transformant, an antibody recognizing the polypeptide, a method for producing a type 1 sugar chain-containing sugar chain using the polypeptide, and a glycoconjugate containing the sugar chain, and a transformation having the recombinant vector.
- the present invention relates to a method for producing a type 1 sugar chain-containing sugar chain and a complex carbohydrate containing the sugar chain using a body.
- Glycans are involved in life phenomena such as development and differentiation and cell recognition, and are thought to be deeply involved in the development and progression of inflammation, cancer, infectious diseases, autoimmune diseases, and many other diseases. [Kibata Yo, Hakomori Senichiro, Nagai Katsu ed., Glycobiology Series I-II, Kodansha, (1993), Glycobiology, 3, 97 (1993)].
- Sugar chains are added to proteins and lipids to exist as glycoproteins, proteoglycans, or glycolipids, and also as oligosaccharides.
- a sugar chain having a Gal3 ⁇ 3GlcNAc structure is called a type 1 sugar chain, and constitutes a skeletal sugar chain of a Lewis blood group antigen or a cancer-associated sugar chain antigen.
- Lewis blood group antigens include Lewis a sugar chain (Gal? 1-3 (Fuca 4) GlcNAc) and Lewis b sugar chain [Fuc
- GlcNAc sialyl Lewis c-glycans
- NeAc a2-3Gal-l-3GlcNAc sialyl Lewis c-glycans
- gastrointestinal cancers such as colorectal cancer and Tengler cancer.
- adhesion molecule selectin E-, P-, and L-selectin
- sugar chain ligands sialyl Lewis X sugar chain or its related sugar chain
- Sialyl Lewis X sugar chain QieuAc 2-3-Gal 4 (Fuc 3) GlcNAc] is a structural isomer of Sialyl Lewis a sugar chain, which binds to selectin. Have been. In addition, there is a report that the expression level of sialyl Lewis a sugar chain in colorectal cancer and penile cancer is correlated with poor prognosis of the cancer.
- the Gal / trit 3GlcNAc structure is synthesized by GlcNAc 51,3-galactosyltransferase. So far, three GlcNAc-l, 3-galactosyltransferase (? 3Gal-Tl,? 3Gal-T2, and? 3GalT3) genes have been cloned and their respective genes have been cloned. The acceptor substrate specificity of the enzyme has been analyzed [JP-A-6-181759, J. Biol. Chem., 273, 58-65 (1998), J. Biol. Chem., 273, 433-440 (1998), J. Biol. Chem., 273, 12770-12778 (1998)]. In addition, another /?
- T 4 synthesizes gangliosides GA1, GM1, or GDlb, but does not synthesize the Gal3G3NAc structure.
- GlcNAc 51,3-galactosyltransferase involved in the synthesis of cancer-related sugar chains, sialyl Louis a-glycan and sialyl Lewis c-glycan can be identified in gastrointestinal cancers such as colorectal cancer and Teng's carcinoma. It is presumed that a more accurate diagnosis of cancer can be made by examining the expression level of the enzyme or the enzyme gene. In addition, suppression of the enzyme activity or transcription and translation of the enzyme gene is expected to enable suppression of cancer metastasis. However, the enzyme or the enzyme gene has not been identified so far.
- GlcNAc 51,3-galactosyltransferase from colon cancer cell line Colo205 Although the enzyme has been partially purified, isolation of the enzyme, determination of the amino acid sequence of the enzyme, and isolation of the enzyme gene have not been completed [J. Biol. Chem., 262, 15649-15658 ( 1987), Archi. Biochem. Biophys. 270, 630-646 (1989), Archi. Biochem. Biophys. 274, 14-25 (1989)].
- a sugar chain having a strong binding ability to selectin is useful as a selectin antagonist in the treatment and prevention of inflammation and cancer metastasis. Therefore, it is involved in the synthesis of sialyl Lewis a sugar chains in gastrointestinal cancers such as colorectal cancer and renal carcinoma, etc.
- 1,3—Galactose transferase is an efficient method for selectin antagonism. It is estimated that it can be used for synthesis.
- the present invention utilizes a polypeptide having a novel /? 1,3-galactosyltransferase to improve pharmaceutical products such as anti-inflammatory, anti-infectious disease, or cancer metastasis, foods such as dairy products, and protein improvement. And a method for diagnosing diseases such as cancer.
- the present invention relates to the following (1) to (47).
- the polypeptides having novel /? 1,3-galactosyltransferase activity of the present invention include not only colon cancer cells expressing sialyl Lewis a sugar chains but also sialyl Lewis c sugar chains and Lewis a sugar chains. It is also present in gastrointestinal cancer cells such as colon cancer cells and renal cancer cells expressing type 1 sugar chains such as Lewis b sugar chains.
- polypeptides having a 1,3-galactosyltransferase activity involved in the efficient synthesis of type 1 sugar chains present in these digestive cancer cells are also the polypeptides of the present invention.
- the polypeptide of the present invention is a novel 31,3-galactosyltransferase which is different from the known / 3Gal-Tl, 53Gal-T2 and / 3Gal-T3.
- Gal5tri3GlcNAc structure comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide of the above (a) or (b), and having the polypeptide.
- Possible /? Polypeptides having 1,3-galactosyltransferase activity are described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as Molecular 'Cloning No. 2). Edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter abbreviated as' protocols in 'molecular' biology), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl.
- the number of amino acids to be deleted, substituted or added is not particularly limited, it is a number that can be deleted, substituted or added by a well-known method such as the site-directed mutagenesis described above.
- the number is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
- the amino acid sequence of SEQ ID NO: 1 and BLAST CJ. Ol. Biol., 215, 403 (1990)] and FAS TA CMethods in Enzymology, 183, 63-98 (1990)], etc. The calculation method and method for defining% homology are described. It is preferred that the homology is at least 60% or more, usually 8 °% or more, particularly 95% or more.
- the amino acid sequence of the above-mentioned polypeptide (a) or (b) has an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and is capable of synthesizing a Gal ⁇ -3GlcNAc structure.
- Polypeptides having galactosyltransferase activity are novel /? 1,3 different from known /? 3Ga1-T1, 53Ga1-T2 and /? 3Ga1-T3.
- It is a galactose transferase.
- the ⁇ 1, 3-galactosyltransferase activity is the activity of transferring galactose to the ⁇ -acetylglycosamine residue present at the non-reducing terminal of the sugar chain by a 1,3 bond.
- the polypeptide according to (2) is the polypeptide according to (2).
- the ⁇ 1,3-galactosyltransferase activity is due to the N-acetylglucosamine residue or N-acetyl present at the non-reducing end of GlcNAc? 3Gal? 4Glc.
- the polypeptide according to the above (1) or (2) which has an activity of transferring galactose to glucosamine monosaccharide by 1,3 bonds.
- DNA selected from the group consisting of the following (a), (b), (c) and (d):
- DNA that hybridizes under stringent conditions refers to a DNA selected from the group consisting of the above DNAs (a), (b) and (c) as a probe, Seesion method, plaque 'DNA that can be obtained by using the hybridization method or the Southern plot hybridization method.Specifically, DNA derived from colonies or plaques is immobilized.
- hybridizable DNA examples include those calculated using BLAST [J. Mol. Biol., 215, 403 (1990)] and FASTA Methods in Enzymology, 183, 63-98 (1990)]. (Calculation means and methods for defining% homology are described), at least 60% homology with the nucleotide sequence of DNA encoding the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 DNA having homology, preferably DNA having homology of 80% or more, more preferably DNA having homology of 95% or more.
- Polypeptides capable of synthesizing the Gal / triol 3GlcNAc structure encoded by the DNA and having a 1,3,3-galactosyltransferase activity are known /? 3Ga1-T1,? 3Ga1-T2 It is a novel /? 1,3-galactosyltransferase that is different from /? 3Ga1-T3.
- transformant is a transformant selected from the group consisting of a microorganism, an animal cell, a plant cell, an insect cell, a non-human transgenic animal, and a transgenic plant. Convertible.
- Animal cells are mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CH0 cells, BHK cells, African green monkey kidney cells, Namalwa cells, Namalwa KJM-1 cells, human embryos Kidney cells and human leukemia cells
- insect cell is an insect cell selected from the group consisting of an ovary cell of Spodoptera f rugiperda, an ovary cell of Trichoplusiani, and an ovary cell of Ryco.
- a transformant having a recombinant DNA obtained by incorporating a DNA encoding the polypeptide according to any one of the above (1) to (4) into a vector is cultured in a culture medium.
- a method for producing the polypeptide comprising: producing and accumulating the polypeptide in the culture; and collecting the polypeptide from the culture.
- an oligosaccharide having an N-acetylglucosamine residue at the non-reducing end ii) an acceptor substrate selected from a complex carbohydrate having an N-acetylglucosamine residue at the non-reducing end, and
- an acceptor substrate selected from the group consisting of glycoconjugates having a glucose residue at the non-reducing end of the sugar chain, and
- a transformant selected from the group consisting of the transformant derived from the microorganism, animal cell, plant cell and insect cell according to (9) above is cultured in a culture medium, and the galactose is added to the culture.
- Tose produces / accumulates N-acetylglycosamine, N-acetylglycosamine residue, sugar chain having a structure added to glucose or glucose residue, or complex carbohydrate containing the sugar chain at 1,3 bonds
- the non-human transgenic animal described in the above (9) is bred, wherein galactose is N-acetylglucosamine, N-acetylglucosamine residue, glucose or glucose having 51,3 bonds in the animal.
- galactose is N-acetylglucosamine, N-acetylglucosamine residue, glucose or glucose having 51,3 bonds in the animal.
- the complex carbohydrate is a complex carbohydrate selected from the group consisting of glycoproteins, glycolipids, proteoglycans, glycopeptides, lipopolysaccharides, peptidoglycans, and glycosides in which sugar chains are bonded to steroid compounds, etc.
- a diagnostic agent for cancer or cancer metastasis comprising the antibody according to (32).
- a method for screening a compound that alters the activity of the polypeptide which comprises contacting the polypeptide according to any one of the above (1) to (4) with a test sample. .
- a cell expressing the polypeptide according to any one of (1) to (4) above is brought into contact with a test sample, and an anti-Sialyl Lewis a antibody, an anti-Lewis a antibody, an anti-Lewis b antibody or an anti-Sialyl Lewis using a c antibody to measure the content of a sialyl Lewis a sugar chain, a Lewis al chain, a Lewis b sugar chain or a sialyl Lewis c sugar chain, characterized in that the expression of a compound encoding the polypeptide is fluctuated. Screening method.
- a cell expressing the polypeptide of any one of the above (1) to (4) is brought into contact with a test sample, and the polypeptide content is reduced using the antibody of the above (32).
- Methods for preparing a cDNA library include Molecular Cloning 2nd Edition, Current 'Protocols'in' Molecular Biology Supplement 1-38, A Laboratory Manual, 2nd Ed. (1989), DNA Clonin 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995), or other commercially available kits, such as Superscript 'plasmid' system for 'cDNA synthesis' and 'plasmid' Using the Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Gibco BRL)) ⁇ Zap-cDNA-Synthesis' kit (ZAP-cDNA Synthesis Kit, Straugene) And the like.
- Any phage vector, plasmid vector, or the like can be used as a closing vector for preparing a cDNA library as long as it can replicate autonomously in the E. coli K12 strain.
- ZAP Express (Stratagene, Strategies, each other, 58 (1992)), pBluescript II SK (+) CNucleic Acids Research, 17, 9494 (1989)), ZAP II
- E. coli LE392 is transformed to prepare a cDNA library.
- pBluescript II SK (+), pBluescript SK (-) (all manufactured by Stratagene), pDIRECT (Nucleic Acids Research, 18, 6069 (1990)), pCR-ScriptAmpSK (+) (Stratagene, Strategies , 5, 6264 (1992)], pT7Blue ( ⁇ & 611 companies Ltd.], (3 ⁇ 411 [Inbitorojen Inc., 8101 ⁇ 1,111,010, 9, 657 (1991)], pCR-TRAP [Genehunter Co., Ltd.], ⁇ ⁇ (5 , ⁇ 3, company).
- Examples of the plasmid containing DNA of SEQ ID NO: 2 include pAMo-3GT5 and pBS-3GT5 (FERM BP-6645) described in Examples described later.
- any vector can be used as long as it can incorporate the cDNA and can be expressed in animal cells, for example, pcDNAI / Amp, pcDNAI, pCD8 (all commercially available from Funakoshi), pAGE107 (Japanese Patent Publication No. 3-22979, Cytotechnology, 3, 133 (1990)), pREP4 (Invitrogen), pAGE103 (J. Biochem., 101, 1307 (1987)), pAMo, pAMoA (J. Biol. Chem. , 268, 22782-22787 (1993) s aka pAMoPRSA (Japanese Unexamined Patent Publication No. 05-336963), pAS3-3 (Japanese Unexamined Patent Publication No. 2-227075), and the like.
- the obtained transformed cells are cultured by a conventional method.
- antibiotics such as kanamycin, penicillin and streptomycin may be added to the medium during the culturing.
- the target DNA is also prepared by chemically synthesizing DNA encoding the polypeptide. be able to. Chemical synthesis of DNA is performed using a DNA synthesizer manufactured by Shimadzu Corporation using the thiophosphite method, a Perkin 'DNA synthesizer model 1392 manufactured by Elma Inc. using the phosphoramidite method, etc. be able to.
- the above-mentioned oligonucleotides in which the melting temperature (Tm) and the number of bases of both do not extremely change are preferable.
- Specific examples include oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 20 and 21 and the like.
- oligonucleotide derivative examples include an oligonucleotide derivative in which a phosphate ester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide in which an N 3, -P 5 ′ phosphonate bond is an N 3, -P 5 ′ phosphor.
- polypeptide of the present invention Production of novel /? 1,3-galactosyltransferase polypeptide (hereinafter also referred to as polypeptide of the present invention)
- polypeptide of the present invention can be prepared by the methods described in Molecular 'Cloning 2nd Edition, Current' Protocols' in 'Molecular' Biology Supplement 1 to 38, etc. DNA can be expressed and produced in host cells.
- DNA fragment of an appropriate length containing a portion encoding the polypeptide is prepared.
- DNA is prepared by substituting the nucleotide sequence of the portion encoding the polypeptide so that the nucleotide sequence becomes an optimal codon for expression in the host. The DNA is useful for improving the production rate of the polypeptide.
- a recombinant DNA (recombinant vector) is prepared by inserting the DNA fragment or the full-length DNA into the downstream of a promoter in an appropriate expression vector. By introducing the recombinant vector into a host cell suitable for the expression vector, a transformant producing the polypeptide of the present invention can be obtained.
- any prokaryotic cell, yeast, animal cell, insect cell, plant cell, or the like can be used as long as it can express the gene of interest.
- an animal individual or a plant individual can be used.
- the expression vector is capable of autonomous replication in the above host cells, or can be integrated into the chromosome, and contains a promoter at a position suitable for transcription of the novel /? 1,3-galactose transferase gene. Is used.
- Expression base Kuta one, for example, (commercially available from both Beri down Gar Mannheim) P BTrp2, pBTacK pBTac2, pKK233- 2 ( Pharmacia Co.), pSE280 (In Bitorojen Co.), pGEMEX- 1 [Promega (Promega) Co. PQE-8 (QIAGEN), pKYPIO (JP-A-58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSAl CAgric. Biol. Chem., 53, 277 (1989)), pGELl [Proc. Natl. Acad. Sci.
- a plasmid in which the distance between the Shine-Dalgarno sequence, which is the ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases) is used. Preferably, it is used.
- host cells include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., for example, Escherichia col i X-Blue Escherichia coli XL2- Blue, Escherichia coli DHU Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL DE3) pLysS,
- Escherichia coli brain 74 (DE3), Escherichia coli HMS174 (DE3) pLysS, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens AT, bacterium ATCC, Brevibacterium ammonia bacterium ATCC bacterium ATCC ATCC14067, Corynebacterium glutamicum ATCC13869,
- Any method for introducing the recombinant vector can be used as long as it is a method for introducing DNA into the above host cells.
- electoporation method CNucleic Acids Res., 16, 6127 (1988)], calcium ion Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (63-248394), Gene 17, 17, 107 (1982), and Molecular & General Genetics, 168, 111. (1979).
- host cells examples include yeast strains belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schizinomyces, Pichia, and the like.Specifically, Saccharomyces cerevisiae,
- any method for introducing DNA into yeast can be used.
- electroporation Methods. EnzymoL, 194, 182 (1990)
- USA 84, 1929 (1978)
- the lithium acetate method J. Bacteriol., 153, 163 (1983)
- any promoter that can be expressed in animal cells can be used.
- the enhancer of the IE gene of human CMV may be used together with the promoter.
- Host cells include mouse myeloma cells, rat 'myeloma cells', mouse' hyperidoma cells, Chinese hamster cells CH0 cells, BHK cells, African green monkey kidney cells, and human cells. Examples include Namalwa cells or Najnalwa KJM-1 cells, human fetal kidney cells, human leukemia cells, HBT5637 (JP-A-63-299), and human colon cancer cell lines.
- any method can be used as long as it is a method for introducing DNA into animal cells.
- the recombinant gene transfer vector and the baculovirus are co-introduced into insect cells to obtain a recombinant virus in the insect cell culture supernatant, and then the recombinant virus is obtained.
- Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacII (all manufactured by Invitrogen) and the like.
- insect cells ovary cells of Spodoptera frugiperda, ovary cells of Trichoplusia, cultured cells derived from silkworm ovary, and the like can be used.
- Spodoptera frugiperda ovarian cells include Sf9 and Sf21 (Baculovirus 'Expression' Vector Vector 'Laboratory 1' manual), etc.
- Ovarian cells of Trichoplusia ni include High 5, BTI-TN-5B1-4 (manufactured by Invitrogen), and Bombyx mori N4 and the like.
- Methods for co-transferring the above-described recombinant gene into insect cells and the above baculovirus into insect cells for preparing a recombinant virus include, for example, calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), and Lipofexion method. [Proc. Natl. Acad. Sci. USA, M, 7413 (1987)].
- DNA can be introduced into insect cells using the same method as that used to introduce DNA into animal cells.
- the polysaccharide is prepared according to a known method [tissue culture, 20 (1994), tissue culture, 21 (1995), Trends in Biotechnology, 15, 45 (1997)].
- the expression vector include Ti plasmid and tobacco mosaic virus vector.
- any promoter can be used as long as it can be expressed in a plant cell, and examples thereof include the 35S promoter of cauliflower mosaic virus (CaMV) and the geneactin 1 promoter.
- the expression efficiency of the gene can also be increased by inserting the maize alcohol dehydrogenase gene intron 1 between the promoter and the gene to be expressed.
- Examples of the host cell include plant cells such as potato, tobacco, corn, rice, rape, soybean, tomato, carrot, wheat, barley, rye, Alfalpha, flax and the like.
- any method for introducing DNA into plant cells can be used.
- Agrobacterium Japanese Patent Application Laid-Open Nos. -78080, W094 / 00977
- an electoral-portation method Japanese Patent Application Laid-Open No. 60-251887
- a method using a particle gun Japanese Patent No. 2517813
- Japanese Patent No. 2517813 Japanese Patent No. 2517813
- the cells or organs of the plant into which the gene has been introduced can be cultured in large quantities using a jar fermenter.
- culture medium use commonly used Murashige 'and' squeeg (MS) medium, white (White) medium, or medium supplemented with plant hormones such as auxin, cytokinin, etc. be able to.
- MS Murashige 'and' squeeg
- White white
- plant hormones such as auxin, cytokinin, etc.
- the cultivation is usually performed at pH 5 to 9, 20 to 40 ° C for 3 to 60 days.
- antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.
- a plant into which the gene has been introduced (transgenic plant) can also be created by redifferentiating the plant cell into which the gene has been introduced.
- Animal polypeptides can also be used to produce the polypeptides of the present invention.
- An animal into which a gene has been introduced according to a known method [American Journal of Clinical Nutrition, 63, 639S (1996), American Journal of Clinical Nutrition, 63, 627S (1996), Bio / Technology, 9, 830 (1991)].
- the polypeptide of the invention can be produced therein.
- promoter Any type of promoter can be used as long as it can be expressed in animals.
- mammary gland cell-specific promoters such as Hicasein Promo — Yuichi, Casein Promo Yuichi,? Globulin promoter overnight, whey acidic protein promoter evening, etc. are preferably used.
- a transformant derived from a microorganism, animal cell, or plant cell having a recombinant vector into which the DNA encoding the polypeptide of the present invention has been incorporated is cultured according to a conventional culture method to produce the polypeptide.
- the polypeptide can be produced by accumulating and collecting the polypeptide from the culture.
- the transformant is an animal or plant individual
- the animal is bred or cultivated according to a conventional method to produce and accumulate the polypeptide, and the polypeptide is collected from the animal or plant individual.
- Polypeptides can be produced.
- a non-human transgenic animal carrying the DNA of the present invention is bred, and a polypeptide having a novel /? 1,3-galactose transferase activity encoded by the recombinant DNA is obtained. Is produced and accumulated in the animal, and the polypeptide is collected from the animal, whereby a polypeptide having a novel /? 1,3-galactose transferase activity can be produced. Examples of the production-accumulation site in the animal include milk, eggs, and the like of the animal.
- a transgenic plant having the DNA of the present invention is cultivated, and a polypeptide having a novel /?
- 1,3-galactose transferase activity encoded by the recombinant DNA is added to the plant.
- a polypeptide having a novel /? 1,3-galactose transferase activity can be produced.
- the transformant for producing a polypeptide of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as a yeast
- these transformants of the present invention are cultured in a medium, and the polypeptide of the present invention is contained in a culture.
- the polypeptide of the present invention can be produced by producing and accumulating the peptide and collecting it from the culture.
- the method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
- a culture medium for culturing a transformant obtained using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the organism. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture C.
- the carbon source may be any one that can be assimilated by each microorganism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, propionic acid, etc. Alcohols such as organic acids, ethanol, and propanol can be used.
- Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other inorganic acids, ammonium salts of organic acids, and other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, and corn starch. , Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermentation cells and digests thereof can be used.
- potassium monophosphate potassium monophosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.
- Cultivation is performed under aerobic conditions such as shaking culture or deep aeration and stirring culture.
- the culture temperature is preferably 15 to 40 ° C, and the culture time is usually 5 hours to 7 days.
- the pH is maintained at 3.0 to 9.0. Adjustment of pH is performed using an inorganic or organic acid, alkali solution, urea, calcium carbonate, ammonia or the like.
- antibiotics such as ampicillin and tetracycline may be used if necessary. It may be added to the medium.
- an inducer may be added to the medium, if necessary.
- an inducer may be added to the medium, if necessary.
- isopropyl-15-D-thiogalactoviranoside or the like is used.
- a medium for culturing the cell may be a commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967) ], Eagle's MEM medium [Science, 122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] or A medium obtained by adding fetal bovine serum or the like to these mediums is used.
- Cultivation is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C, and 5% C ⁇ 2 .
- antibiotics such as kanamycin, penicillin, and streptomycin may be added to the medium during the culturing.
- the medium for culturing the cell may be a commonly used TNM-FH medium (Pharmingen), Sf-900II SFM A culture medium (manufactured by Gibco BRL), ExCell 400, ExCell 405 (all manufactured by JRH Biosciences), Grace's Insect Medium (Nature, 195, 788 (1962)) and the like can be used.
- Gene expression methods include, besides expressing the full-length polypeptide,
- Glycosyltransferases generally have a type 2 membrane protein topology, an N-terminal cytoplasmic region consisting of a few to several tens of amino acids, a membrane-binding region having a highly hydrophobic amino acid sequence, and a few to a few tens of amino acids. It consists of a stem region and most of the remaining C-terminal portion, including the catalytic region. Most of the remaining C-terminal portion, including the stem and catalytic regions, appears to be exposed in the Golgi lumen.
- the boundary between the stem region and the catalytic region can be determined experimentally by preparing a polypeptide from which the N-terminus has been deleted and examining how far the deletion will result in loss of activity. On the other hand, by comparing the amino acid sequence with a similar glycosyltransferase that has knowledge of the stem region and the catalytic region, the stem region and the catalytic region can also be predicted.
- the structure of the novel /? 1,3-galactosyltransferase of the present invention has the same structure as other glycosyltransferases.
- the polypeptide of the present invention having the amino acid sequence shown in SEQ ID NO: 1, at least a cytoplasmic region consisting of 7 amino acids at the N-terminal, followed by a hydrophobic-rich membrane-binding region consisting of 19 amino acids, It consists of a stem region consisting of 4 amino acids, and most of the remaining C-terminal portion including the catalytic region.
- a polypeptide comprising the 31st to 310th amino acid sequence is considered to contain a catalytic region.
- the partial polypeptide containing the full-length polypeptide or a region having a 1,3-galactosyltransferase activity is described in Molecular Cloning Second Edition in addition to direct expression. It can also be expressed as a secreted protein or a fusion protein according to the method or the like.
- the proteins to be fused include galactosidase, protein A, the IgG binding region of protein A, chloramphenicol acetyltransferase, poly (Arg), Poly (G lu), protein G, maltose binding protein, glutathione S-transferase, polyhistidine chain (His-tag), S peptide, DNA binding protein domain, Tac antigen, thioredoxin, green ' Fluorescent protein and any antibody epitope [Akio Yamakawa, Experimental Medicine, 13, 469-474 (1995)].
- the method for producing the polypeptide of the present invention includes a method of producing the polypeptide in a host cell, a method of secreting the polypeptide outside the host cell, and a method of producing the polypeptide on the host cell outer membrane.
- the method can be selected by changing the structure of the polypeptide.
- the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
- an ordinary enzyme isolation / purification method can be used. For example, when the polypeptide of the present invention accumulates in a dissolved state in the cells of the transformant for producing the polypeptide of the present invention, the cells in the culture are collected by centrifuging the culture. After washing the cells, the cells are crushed with an ultrasonic crusher, a French press, a Mantongaulin homogenizer, a Dynomill or the like to obtain a cell-free extract.
- Galactose is a 1,3-linked N-acetylglucosamine, N-acetylglucosamine residue, a sugar chain having a structure added to glucose or a glucose residue, and a complex carbohydrate containing the sugar chain.
- a structure in which galactose is added to the N-acetylglycosamine residue via /? 1,3 bonds As a sugar chain containing a structure, a sugar chain containing a sialyl Lewis a structure, a sugar chain containing a sialyl Lewis c structure, a sugar chain containing a Lewis a structure, a sugar chain containing a Lewis b structure, and Gal Gal 3Gal n -A sugar chain containing a 3GlcNAc structure, Gal 1-3 (Fuc 2) Gal? 1-3-A sugar chain containing a 3GlcNAc structure, GalNAc al-3 (Fuc al-2) Gal ⁇ l-3GlcNAc structure And the like.
- a surfactant or an organic solvent may be added to the aqueous medium as needed.
- the surfactant include nonionic surfactants such as polyoxyethylene and octane decylamine (for example, Nimeen S-215, manufactured by NOF Corporation), cetyltrimethylammonium, bromidealkyldimethyl, benzylammoniumchromate.
- glycoconjugate having an N-acetylglycosamine residue at the non-reducing end of the sugar chain a glycoconjugate containing a sugar chain having any one of the structures of the oligosaccharide at the non-reducing end of the sugar chain And complex carbohydrates containing an asialo-galacto complex-type N-linked sugar chain.
- Animals such as egrets, goats, rats, mice, and hamsters can be used for the administration.
- Serum is obtained from a non-human mammal whose serum has a sufficient antibody titer against the antigen used for immunization, and a polyclonal antibody can be separated and purified from the serum by the following method.
- Rats whose sera showed a sufficient antibody titer against the partial polypeptide of the polypeptide of the present invention used for immunization are used as a source of antibody-producing cells.
- the spleen is shredded in a MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
- a monoclonal antibody can be purified and obtained in the same manner as in the polyclonal method.
- the antibody subclass is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit.
- the antibody class refers to the antibody isotype, and in humans, includes IgG, IgA, IgM, IgD, and IgE.
- the subclass is an isotype within a class, and includes IgGl, IgG2a, IgG2b, IgG3 in mice and IgGl, IgG2, IgG3, and IgG4 in humans.
- the human chromosomal gene encoding the polypeptide of the present invention was identified by comparing the sequence of the human cDNA encoding the polypeptide of the present invention with the sequence of the human chromosomal gene registered on the database.
- the structure of the gene can be clarified. If a chromosomal gene sequence that matches the sequence of the cDNA is registered, the sequence of the chromosome gene is compared with the sequence of the chromosome gene to obtain the promoter region of the chromosomal gene encoding the polypeptide of the present invention.
- the exon and intron structures can be determined.
- diagnosis of disease and prediction of prognosis can be performed by examining the Fuc-TII I polymorphism.
- the polymorphism analysis of the present gene can be performed using the gene sequence information of the present gene. Specifically, genetic polymorphisms can be analyzed using the Southern plot method, direct sequence method, PCR method, DNA chip method, etc. [Clinical test, 42, 1507-1517 (1998), clinical test, 42, 1565-1570 (1998)]. (6) Use of the polypeptide of the present invention
- polypeptide of the present invention can be screened for a compound that enhances or inhibits the activity of the polypeptide by the method (a) of (8) below.
- Examples of the immunological detection method of the polypeptide of the present invention include an ELISA method using a microplate, a fluorescent antibody method, a Westin blot method, and an immunohistological staining method.
- the immunological assay Sanditsuchi ELISA method E Pi Bok one flop using two different monoclonal antibodies of the antibodies that react with polypeptides of the present invention in the liquid phase, radioactive isotopes, such as 126 1 And a radioimmunoassay method using the polypeptide of the present invention and an antibody recognizing the polypeptide of the present invention.
- the above detection or quantification method can be used for diagnosis of colorectal cancer, Teng's cancer, and the like.
- the antibody of the present invention can be used as a medicament, for example, as a therapeutic agent for diseases such as colorectal cancer and thyroid cancer.
- the pharmaceutical containing the antibody of the present invention can be administered alone as a therapeutic agent, it is usually mixed with one or more pharmacologically acceptable carriers to give a pharmaceutical preparation. It is desirable to provide as a pharmaceutical preparation manufactured by any method well known in the technical field of the invention.
- Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
- Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.
- liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, sesame oil, olive oil, soybean oil, etc.
- oils preservatives such as p-hydroxybenzoic acid esters, flavors such as strawberry flavor and ⁇ permint as additives.
- Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, lubricants such as magnesium stearate, sugar, polyvinyl alcohol, It can be produced using a binder such as hydroxypropylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.
- the dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 // to 8111/1 ⁇ per adult per day.
- the novel /? 1,3-galactosyltransferase polypeptide of the present invention can be used in gastrointestinal cancer cells such as colon cancer cells and kidney cancer cells to produce sialyl Lewis a sugar chains, sialyl Lewis c sugar chains, Lewis a sugar chains, Because it is involved in the synthesis of type 1 sugar chains such as Lewis b sugar chains, use a compound that enhances or inhibits the activity of the polypeptide to increase or decrease the amount of type 1 sugar chains synthesized in cells Is possible.
- a compound that promotes or suppresses the transcription process of a gene encoding the polypeptide or the translation process from a transcript to protein controls the expression of the polypeptide, and suppresses the type 1 sugar in a cell. It is possible to control the amount of chain synthesis.
- the above compounds can be obtained by the following methods (a) to (e).
- Examples of the measurement method using the above antibody include a ELISA method using a microplate, a fluorescent antibody method, a Western blot method, and a detection method using immunohistochemical staining.
- the amount of the polypeptide in the cells is determined by the method of (4). Measurement is performed using the antibody of the present invention described above, and a compound having an activity of increasing or decreasing the amount of the polypeptide is selected and obtained.
- a detection method using an ELISA method using a microplate for example, a detection method using an ELISA method using a microplate, a fluorescent antibody method, a Western plot method, immunohistological staining, or the like can be mentioned.
- a plasmid in which a DNA obtained by linking the repo overnight gene was incorporated downstream of the promoted night obtained in (4) above was prepared by a known method, and the plasmid was added to the animal cell described in (2) above.
- the repo genes include, for example, the chloramphenicil 'acetyltransferase gene, the? -Glucuronidase gene, the galactosidase gene, the lactate gene, the luciferase gene, the equorin gene or the green fluorescent protein. GFP) gene and the like.
- the target gene on a chromosome in a non-human animal for example, an embryonic stem cell (embryonic stem cell) such as a sea lion, a sheep, a goat, a bush, a horse, a chicken, or a mouse.
- embryonic stem cell embryonic stem cell
- the DNA encoding the polypeptide of the present invention is inactivated or inactivated by a known homologous recombination technique (for example, Nature, 326, 6110, 295 (1987), Cell, 51, 3, 503 (1987), etc.). Mutant clones can be created that have been replaced with any sequence [eg, Nature, 350, 6315, 243 (1991)].
- a chimera comprising an embryonic stem cell clone and normal cells by a technique such as an injection chimera method or a collective chimera method. Individuals can be created.
- the polypeptide of the present invention on the chromosome is Mutations can be introduced at any position in the DNA to be loaded. For example, by introducing mutations such as base substitutions, deletions, and insertions into the translation region of the DNA encoding the polypeptide of the present invention on the chromosome, the activity of the product can be changed. Further, by introducing a similar mutation into the expression control region, it is possible to modify the degree, timing, tissue specificity, etc. of the expression. Furthermore, the expression timing, expression site, expression amount, and the like can be more positively controlled by combination with the Cre-- ⁇ system.
- Examples of such cases include the use of promoters expressed in a specific region of the brain and deletion of the target gene only in that region [Cell, 87, 7, 1317 (1996)] and Cre. It has been known that an organ-specific deletion of a target gene at an intended time using an adenovirus that expresses [Science, 278, 5335, (1997)].
- the expression of the DNA encoding the polypeptide of the present invention on the chromosome can be controlled at any time or in any tissue, or any insertion, deletion, or substitution can be performed in the translation region or expression control region. It is possible to produce a knockout non-human animal having the above.
- Such a knockout non-human animal can induce symptoms of various diseases caused by the polypeptide of the present invention at any time, any degree, or any site.
- the knockout non-human animal of the present invention becomes an extremely useful animal model in the treatment and prevention of various diseases caused by the polypeptide of the present invention.
- it is very useful as a model for evaluation of therapeutic and prophylactic agents, functional foods, health foods, etc.
- Fig. 1 A in Fig. 1 shows the results of measuring the expression levels of type 1 sugar chains (sialyl-sugar a-sugar, Lewis-a-sugar, and Lewis-b-sugar) in various human cancer cell lines. .
- type 1 sugar chains sialyl-sugar a-sugar, Lewis-a-sugar, and Lewis-b-sugar
- Analysis was performed using ACS.
- the +10, +10, 10, Sat, and I are shown in order of strong reactivity with each antibody. One indicates that there was no reactivity with the antibody.
- NT means not analyzed.
- FIG. 1 shows the results of the quantitative PCR method using human 3Ga1-T1, human-3Ga1-T2, human-3Ga1-T2 in various human cancer cell lines.
- FIG. 3 is a view showing the results of quantifying the amounts of transcripts of 3Ga1— ⁇ 3 and human 3Gal—T4. The amount of each transcript of 1,3-galactosyltransferase gene in various cell lines is considered to be expressed to the same extent in all cells. When the amount of transcript of? -Actin is 1000 It was displayed as a relative value of
- FIG. 2 shows the construction process of Plasmid pBS-3GT5.
- Fig. 3 A in Fig. 3 shows the appearance of Namalwa cells (Namalwa (mock)) transfected with control plasmid (pAMo) or human?
- An indirect fluorescent antibody was obtained from Namalwa cells (Namalwa-3GT5) transfected with 3G a1-T5 expression plasmid (pAM0-3GT5) using anti-Sialyl Lewis c sugar chain antibody (DU-PAN-2).
- FIG. 3 is a view showing the results of analysis using FACS after staining. The shaded histogram is the result when A-PBS was used instead of DU-PAN-2.
- 4A shows the results of quantifying the amount of human 3 Ga1-T5 transcript in various human cancer cell lines using quantitative PCR.
- the amount of human 33Gal-T5 transcripts in various cell lines is considered to be expressed to the same extent in all cells. It was displayed as a relative value when it was done.
- FIG. 4B shows the results of examining the expression of CA19-9 antigen-containing protein in various human cancer cell lines by Western blotting analysis.
- FIG. 5 is a diagram showing the results of quantifying the amount of human 3Ga1-T5 transcript in various human tissues using the quantitative PCR method. The amount of transcript of human ⁇ 3Ga1-T5 in each tissue is considered to be expressed to the same extent in all cells.-The amount of transcript of actin is expressed as a relative value when the amount of transcript is set to 1000. did.
- FIG. 6A is a diagram showing the structure of the human 33 Ga 1-T5 chromosome gene.
- the four exons are squares, and the introns are lines.
- the coding area (open reading frame) is shaded.
- the positions of the primers (si-1, si-2, si-3, si-4) used for the analysis of the isoform of 3G a1—T5c DNA are indicated by arrows.
- FIG. 6B shows the structure of the isoform of human 3Gal-T5c DNA.
- the abundance ratio indicates the expression amount of each isoform in Colo205 cells as a percentage.
- FIG. 3 shows the results of examining the expression levels of human 3 Ga1-T5 cDNA isoforms in Colo205 cells using the ⁇ method.
- isoforms were identified by digestion with the restriction enzymes (XbaI or Bsml) shown in FIG. none means that no restriction enzyme treatment is performed.
- the left lane shows the molecular weight (100 bp ladder).
- Example 1 Measurement of expression level of type 1 sugar chain and expression level of known /? 1,3-galactose transferase gene in various cell lines
- type 1 sugar chains Sialyl Lewis a sugar chain, Lewis a sugar chain, Lewis b sugar chain
- 1,3-galactosyltransferase gene By measuring the expression level of 1,3-galactosyltransferase gene, it is involved in the synthesis of type 1 sugar chains in colon cancer cell lines or rutile cancer cell lines /? 1,3-galactose transfer An attempt was made to identify the enzyme.
- type 1 sugar chain Sialyl Lewis a sugar chain, Lewis a sugar chain, Lewis b sugar chain
- anti-Sialyl Lewis a sugar chain antibody anti-Lewis a sugar chain antibody
- anti-Lewis b sugar chain After staining with a fluorescent antibody using a chain antibody, analysis was performed using FACS (A in FIG. 1).
- transcripts of the known /? 1,3-galactosyltransferase (? 3Gal-Tl, 53Gal-T2,? 3Gal- ⁇ 3, 53Gal-T4) genes in various human cell lines — PCR method [Pro Natl. Acad. Sci. USA., 87, 2725 (1990), J. Biol. Chem., 269, 14730 (1994), JP-A 06-181759, J. Biol. Chem., 273 , 26729 (1998)].
- the amount of each ⁇ 1,3-galactose transferase gene transcript in various cell lines is considered to be expressed to the same extent in all cells.
- the relative value when the amount of --actin transcript is 1000 (B in Fig. 1).
- Cell lines include colorectal cancer cell lines (Colo205, Colo201, SW1116, LS180, HT29, WiDr, HCT-15, SW480, SW620), ligament cancer cell lines (Capan-1, Capan-2), gastric cancer cell lines (KATOII I, MKN45, MKN74), lung cancer cell line (PC-1), neuroblastoma cell line (SK-N-MC, SK-N-SH), lymphoma cell line (Namalwa, Jurkat), prostate cancer cell The strain (PC-3) was used.
- each cell After culturing the above cells in a medium suitable for each cell, each cell is cultured with an anti-Sialyl Lewis a glycan antibody (19-9), anti-Lewis a glycan antibody (7LE), or an anti-Lewis b glycan antibody (Neokokusai Was stained with a fluorescent antibody and analyzed using FACS. The specific method is shown below.
- Each cell (about 1 ⁇ 10 6 ) was placed in a microtube (1.5 ml: manufactured by Eppendorf), and the cells were collected by centrifugation (550 ⁇ g, 7 minutes).
- the cells were washed with 0.191111 of phosphate buffer PBS containing 0.1% sodium azide (A-PBS: 8 g / 1 NaCl, 0.2 g / 1 KC1, 1.15 g / 1 N a 2 HP0 4 (anhydrous), 0. 2 g / 1 KH 2 P_ ⁇ 4, washed with 0.1% sodium azide), said the washing cell A- approximately with PBS 10 ⁇ g / m 1
- the anti-glycan antibody diluted above was added and suspended in 20 ⁇ 1, and reacted at 4 ° C for 1 hour.
- the cells were washed once with 0.9 ml of A-PBS, and then an anti-mouse IgM / IgG antibody (manufactured by Bio-Rad) fluorescently labeled with fluorescein isothiocyanate (FITC) was added to the cells.
- FITC fluorescein isothiocyanate
- the cells were washed once with 0.9 ml of A—PBS, then suspended in 0.6 ml of A—PBS, and the cells were suspended in a fluorescent solution.
- the analysis was performed using Elite's flow cytometer (EPICS Elite Flow Cytometer; manufactured by COULTER). As a control experiment, the same analysis was performed using A-PBS instead of the anti-glycan antibody.
- RNA was extracted from the cell line described in the above (1) by the acid guanidium thiosinate phenol monoclonal form method [Anal. Biochem., 162, 156-159].
- Deoxyribonuclease I (manufactured by Life Technologies) was added to 6 g of each total RNA at 5 units / ml and allowed to react at room temperature for 5 minutes. After the reaction, the enzyme was inactivated by heating at 65 for 15 minutes.
- CDNA was synthesized using the SUPERSCRIPT TM Preamplification System for First Strand cDNA System (Life Technologies). The reaction was performed at 20 il, and the solution after the reaction was diluted 50-fold with water and stored at -80 ° C until use.
- Standards used to create the calibration curve include the human 53Gal-Tl, human 53Gal- ⁇ 2, human-3Gal- ⁇ 3, and human-3Gal- ⁇ 4 cDNAs incorporated into pUC119 or pBluescript SK (-). Plasmid (pUC119-3GT1 ⁇ pBS-3GT2, pBS-3GT3, pBS-3GT4) was cleaved with an appropriate restriction enzyme to cut out each cDNA portion, and converted to linear DNA.
- pUC119-3GTld was prepared by deleting 212 bp between BanII-Ec_2_RV in human-3Gal-Tl cDNA.
- pBS-3GT3d was prepared by deleting 183 bp between StyI and StyI in human 53Gal-T3 cDNA.
- Table 1 shows the nucleotide sequence of each gene-specific primer.
- the base sequence of the 33Gal-T1 gene-specific primer is set to SEQ ID NOs: 4 and 5, and
- the nucleotide sequence of the gene-specific primer is shown in SEQ ID Nos. 8 and 9, the nucleotide sequence of the 53Gal-T4 gene-specific primer is shown in SEQ ID Nos. 10 and 11, and the nucleotide sequence of the? -Actin specific primer is shown in SEQ ID No. 12. , 13 shown.
- 1,3-galactosyltransferase which is involved in the synthesis of type 1 sugar chains in these cell lines, is a novel enzyme.
- mRNA was obtained from human colon cancer cell line Colo205 using Oligotex TM -dT30 super>, an mRNA extraction kit manufactured by Roche. Specific reagents and methods followed the instructions attached to the kit.
- RNA transfer RNA
- tRNA transfer RNA
- 10 mmol / l Tris-HC1 (pH 8.0) and 1 mm 01/1 EDTA Ethylenediamine tetrasodium acetate
- TE buffer a buffer solution
- E. coli LE392 strain [Molecular Cloning 2nd Edition] was transformed by the electroporation method [Nucleic Acids Res., 16, 6127 (1988)], and about 1 million ampicillins were transformed. A transformant having resistance was obtained, and a cDNA library was constructed.
- a plasmid containing cDNA was prepared using the cDNA library (Escherichia coli) and a plasmid preparation kit / plasmid / maxi kit (product number 41031) manufactured by Qiagen.
- RPMI1640 medium containing 10% ⁇ shea fetal serum [7. 5% N a HC 0 3 1/40 volume, 200Mmo 1 / 1% L-glutamine solution (GIBC0) 3%, Penicillin streptova Suspended in 8 ml of RPMI1640 medium (Nissui Pharmaceutical) supplemented with 0.5% of isin solution (GIBCO, 5000 units / ml penicillin, 5000 ⁇ g / m1 streptomycin), and added: (: 0 2 The cells were cultured at 37 ° C. for 24 hours in the incubate.
- the protein concentration of the cell lysate was measured using a micro BCA protein assay reagent kit (PIERCE), and 10 ⁇ g of the protein was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
- the protein on the gel was transferred to an I-bobi Ion PVDF membrane (Millipore) using a Transblot SD cell (Bio-Rad).
- the membrane was treated with 10 g / ml anti-silyl Lewis a antibody (19-9) diluted with blocking solution at room temperature for 2 hours.
- the transformed cells were suspended in a solution [2 Ommo 1/1 HEPES (pH 7.2) 2% TrironX-100], and then sonicated for a short time to prepare a cell lysate.
- the protein concentration of the cell lysate was measured using a micro BCA protein assay reagent kit (PIERCE).
- the activity was measured by using a 10 ⁇ 1 solution of Assay (14 mm 01/1 HE PES (H7.4), 75 zmo 1/1 UDP-Ga 1 (SIGMA), 11 Himo 1/1 MnCl 2,, 0. 01% TrironX- 100 , 25 zmo 1/1 pyridinium Jiruamino sugar chain substrate, 37 ° in the cell lysate] C, 2 hours after the reaction, the product by high performance liquid chromatography (HPLC) Was performed.
- lactone-N-neotetraose (Lacto-N-neotetraose, Gal -4GlcNAc H-3Gal ⁇ 4Glc; hereinafter abbreviated as LNnT) fluorescently labeled with aminopyridine is treated with 5-galactosidase to give a terminal.
- LNnT lactone-N-neotetraose
- 5-galactosidase fluorescently labeled with aminopyridine
- Agaract LN nT is prepared by adding 100 ml of /?-Galactosidase (manufactured by Seikagaku Corporation) to L NnT fluorescently labeled with about 60 nmo 1 of aminoviridine, and reacting at 37 for 16 hours. It was prepared by inactivating 5-galactosidase by heat treatment at 100 ° C for 5 minutes.
- LN n T fluorescently labeled with aminopyridine or lacto-N-tetraose fluorescently labeled with aminoviridine (Lacto-N-tetraose, Gal- 3GlcNAc 3Gal 4Glc; hereinafter abbreviated as LNT).
- LNT lacto-N-tetraose
- LNT lacto-N-tetraose, Gal- 3GlcNAc 3Gal 4Glc; hereinafter abbreviated as LNT.
- LNT lacto-N-tetraose, Gal- 3GlcNAc 3Gal 4Glc
- the Assay solution was treated at 100 ° C. for 3 minutes, centrifuged at 10,000 ⁇ g for 5 minutes to obtain a supernatant, and a part thereof was subjected to HP LC.
- HPLC was carried out using a TSK-ge 1 ⁇ DS-80Ts column (4.6 x 300 mm; manufactured by Tohso Corporation) and 0.02 mol / l ammonium acetate buffer ( ⁇ 4 ⁇ Using 0), elution was performed at 25 ° C and a flow rate of lml / min.
- the product was detected using a fluorescence spectrum photometer FP-920 (manufactured by JASCO Corporation) (excitation wavelength: 320 nm, emission wavelength: 400 nm).
- the product was identified using the standard sugar chain as the elution time as an index.
- the quantification of the product was performed by comparing the fluorescence intensity using lactose that was aminobiliylated as a standard.
- Table 3 shows the relative activity of each /? 1,3-galactose transferase when the activity of human? 3Ga1-T5 is defined as 100.
- the GlcNAc 51,3-galactosyltransferase activity (LNT synthesis activity) of 3 Ga 1—T 5 is similar to that of other GlcNAc ⁇ , 3-galactosyltransferases (human 3Gal — Tl, human 3Gal). — ⁇ 2, human 53 G a1— ⁇ 3). It is known that human 53 Ga1- ⁇ 4 has no GkNAc 51,3-galactosyltransferase activity and has GalNAc 51,3-galactosyltransferase activity.
- human 33 Ga1-T5 was proved to be a GlcNAc-1,3-galactosyltransferase. Also human? The activity of 3 G a1—T 5 is stronger than that of other GlcNAc ⁇ 1,3-galactosyltransferases (human 3 Ga 1- ⁇ 1, human 3Gal—T2, human 3Gal— ⁇ 3) This indicates that it is useful for the synthesis of type 1 sugar chains such as LNT.
- HCT-15 cells HCT-3GT5L and HCT-L5
- 3GT5H colon cancer cell lines
- Colo205, SW1116, HCT-15 stomach cancer cell line
- Capan-2 stomach cancer cell line
- MKN45 gastric cancer cell line
- PC-1 lung cancer cell line
- the expression level of the /? 3 Ga 1 -T 5 transcript in each cell was determined using the method described in Example 1 or Example 2, (7).
- GlcNAc-1,3-galactose transferase activity was measured according to the method described above.
- the expression level of the 3 Ga 1-T 5 transcript was correlated with the activity of GlcNAc-1,3-galactose transferase (LNT synthesis activity).
- LNT synthesis activity the activity of GlcNAc-1,3-galactose transferase.
- the amount of 33Gal-T5 transcript in HCT-3GT5H was about three times that of HCT-3GT5L, but the GlcNAc —1,3-galactosyltransferase activity in HCT-3GT5H (LNT synthesis activity) ) was about three times that of HCT-3GT5L.
- PC-1 expressed a large amount of /? 3Ga1-T1, but no GlcNAc-1,3-galactosyltransferase activity (LNT synthesis activity) was detected.
- MKN45 expressed 3 Ga1-T3 in a large amount, but no GlcNAc-1,3-galactose transferase activity (LNT synthesis activity) was detected.
- the 3 Ga1-T5 transcript was found to be significantly expressed in the stomach (body), stomach (sinus), jejunum, large intestine, and kidney. Some expression was also found in brain, esophagus, kidney and uterus. On the other hand, no expression was found in lung, liver, spleen, adrenal gland, or peripheral lymphocytes.
- sequences of many human chromosome genes of unknown function are registered in the database. Therefore, by comparing the sequence of the human cDNA encoding the polypeptide of the present invention with the sequence of the human chromosomal gene registered in the database, the human chromosomal gene encoding the polypeptide of the present invention can be obtained. It may be possible to identify and clarify its structure. If a chromosomal gene sequence that matches the sequence of the cDNA is registered, the sequence of the cDNA and the sequence of the chromosomal gene are compared to obtain the promoter region of the chromosomal gene encoding the polypeptide of the present invention, exon, and The intron structure can be determined.
- single-stranded cDNA was synthesized using mRNA (1 ig) derived from Colo205 cells as primers and two synthetic DNAs having the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 23 as primers.
- an oligo dC was added to the 3 ′ end of the cDNA using a minaldexoxynucleotidyltransferase, and the synthetic DNA having dG till attached to the kit was subjected to fore-priming primers.
- An array WO 00/50608 PCT / JPOO / 01070—PCR was performed using synthetic DNA having the sequence shown in No. 24 as a reverse primer.
- the PCR was carried out under the conditions that after heating at 97 for 1 minute, 42 cycles of a reaction consisting of 94 at 1 minute, 55 at 1 minute, and 72 ° C for 2 minutes as one cycle.
- the amplified fragment was digested with HindIII and SpeI, and then subcloned between HindIII-SpeI of pBluescript SK (-).
- the nucleotide sequences of the five plasmids thus obtained were determined.
- the transcription start point of the /? 3Ga1-T5 chromosome gene in Colo205 cells was found to be at the 85153rd position on the above-mentioned accession number AF064860. It turned out to be a base.
- the upstream region was a promoter region functioning at least in Colo205 cells.
- the promoter region (including the transcription control region) of the? 3Gal-T5 chromosome gene is estimated to be 5 kb (1 to 5000 of the nucleotide sequence represented by SEQ ID NO: 3) upstream of the transcription start site.
- TF SEARCH transcripiton factor searcn program (Akiyama, Y. , htt:.. // www .rwcp.or jp / 1 ab / pdap 1 / pap i html) 0 , which were analyzed using the
- TATA box Although no TATA box was present upstream of the transcription start site, two CdxA sites, one AP-1 site, and one MZF-l (myeloid zinc finger 1) were found in the 150 bp upstream of the transcription start site. protein) site was presumed to be present.
- Plasmids linked to a reporter gene downstream of this region were introduced into cells expressing? 3Gal-T5, and the promoter region was examined by examining whether or not the repo allele was expressed. Can also be specified.
- the PCR method was used to isolate the / 53 Ga1-T5 cDNA isoform. We examined whether ohms existed.
- the single-stranded cDNA derived from Colo205 cells prepared in Example 1 was subjected to PCR using type II, two synthetic DNAs having the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 25 as primers.
- PCR 94 after heating for 1 minute at 97. (: 1 minute, 55 1 minute, 72 C for 2 minutes), and the cycle was repeated 42 times.
- the amplified fragment was digested with HindIII and then subcloned into HindIII site of pBluescript SK (-).
- the nucleotide sequence of the thus obtained plasmid was determined. As a result, it was found that at least five types of /? 3Gal-T5 cDNA isoforms were present in Colo205 cells (Fig. 6). ).
- the ratio of the expression levels of each isoform was determined. 50% for isoform 1, 50% for isoform 2, and 50% for isoform. Forms 3, 4, and 5 each had less than 1%.
- PCR amplified fragments corresponding to each isoform were identified by the size of the amplified fragments and restriction enzyme treatment (XbaI treatment or Bsml treatment).
- SEQ ID NO: 3 shows the promoter region of the ⁇ 3Ga1_ 35 chromosome gene (including the transcription control region) and the sequence of the /? 3Gal- ⁇ 5 chromosome gene.
- the sequence of the promoter overnight region (including the transcription control region) of the ⁇ 3 Ga 1- ⁇ 5 chromosome gene is l-5000 bp of SEQ ID NO: 3.
- the sequence of the? 3Gal-T5 chromosome gene is from 5001 to 10562 bp of SEQ ID NO: 3.
- Table 4 shows the positions of the exon and the inlet of the 33Gal-T5 chromosome gene using SEQ ID NO: 3.
- the base sequence shown in capital letters indicates the exon portion, and the base sequence shown in lower case letters indicates the intron portion.
- ⁇ 3Ga1_T5 chromosome gene structure (location and sequence of exon and intron regions) and chromosomal location, and / or 3G al— ⁇ Promoter region of chromosome 5 gene The position and sequence of were first identifiable by the present study, which revealed the structure and function of 3G a1—T5 cDNA.
- Table 4 Exon-Intron Binding Site of the ⁇ 3Gal-T5 Chromosome Gene Exon Subrysacceptor Splice Donor in SEQ ID NO: 3
- a novel polypeptide which is involved in the synthesis of a type 1 sugar chain in digestive cancer cells such as colon cancer cells or knee cancer cells, and has a 1,3-galactosyltransferase activity.
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EP00905320A EP1162269A4 (en) | 1999-02-25 | 2000-02-24 | NEW POLYPEPTIDE |
AU26915/00A AU2691500A (en) | 1999-02-25 | 2000-02-24 | Novel polypeptide |
US09/914,152 US7241605B1 (en) | 1999-02-25 | 2000-02-24 | Polypeptide |
CA002365019A CA2365019A1 (en) | 1999-02-25 | 2000-02-24 | Novel polypeptide |
JP2000601172A JP4689047B2 (ja) | 1999-02-25 | 2000-02-24 | 新規ポリペプチド |
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JP11047571A JP2000245464A (ja) | 1999-02-25 | 1999-02-25 | 新規ポリペプチド |
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WO2002079424A2 (en) * | 2001-03-29 | 2002-10-10 | Deltagen, Inc. | TRANSGENIC MICE CONTAINING β3GALT2 GENE DISRUPTIONS |
WO2009028417A1 (ja) | 2007-08-24 | 2009-03-05 | Tokyo Institute Of Technology | 婦人科癌の検出方法 |
RU2623167C2 (ru) * | 2003-01-22 | 2017-06-22 | Гликарт Биотекнолоджи АГ | КОНСТРУКЦИИ СЛИЯНИЯ И ИХ ПРИМЕНЕНИЕ ДЛЯ ПОЛУЧЕНИЯ АНТИТЕЛ С ПОВЫШЕННЫМИ АФФИННОСТЬЮ СВЯЗЫВАНИЯ Fc-РЕЦЕПТОРА И ЭФФЕКТОРНОЙ ФУНКЦИЕЙ |
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ATE408006T1 (de) | 1998-11-13 | 2008-09-15 | Henrick Clausen | Udp-galaktose: beta-i(n)-acetyl-glukosamin beta1, 3 galaktosyltransferasen, beta3gal-t5 |
JP2005065687A (ja) | 2003-01-23 | 2005-03-17 | National Institute Of Advanced Industrial & Technology | β1,3−N−アセチル−D−ガラクトサミン転移酵素タンパク質及びそれをコードする核酸、並びにそれを用いた癌化検定方法 |
CA3149497A1 (en) | 2019-08-09 | 2021-02-18 | Nutcracker Therapeutics, Inc. | Methods and apparatuses for manufacturing for removing material from a therapeutic composition |
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JPH06181759A (ja) * | 1992-12-16 | 1994-07-05 | Kyowa Hakko Kogyo Co Ltd | β1,3−ガラクトシルトランスフェラーゼ |
JPH1156373A (ja) * | 1997-08-27 | 1999-03-02 | Koichi Furukawa | 糖転移酵素遺伝子 |
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- 2000-02-24 AU AU26915/00A patent/AU2691500A/en not_active Abandoned
- 2000-02-24 WO PCT/JP2000/001070 patent/WO2000050608A1/ja active Application Filing
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JPH06181759A (ja) * | 1992-12-16 | 1994-07-05 | Kyowa Hakko Kogyo Co Ltd | β1,3−ガラクトシルトランスフェラーゼ |
JPH1156373A (ja) * | 1997-08-27 | 1999-03-02 | Koichi Furukawa | 糖転移酵素遺伝子 |
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WO2002079424A2 (en) * | 2001-03-29 | 2002-10-10 | Deltagen, Inc. | TRANSGENIC MICE CONTAINING β3GALT2 GENE DISRUPTIONS |
WO2002079424A3 (en) * | 2001-03-29 | 2003-09-04 | Deltagen Inc | TRANSGENIC MICE CONTAINING β3GALT2 GENE DISRUPTIONS |
RU2623167C2 (ru) * | 2003-01-22 | 2017-06-22 | Гликарт Биотекнолоджи АГ | КОНСТРУКЦИИ СЛИЯНИЯ И ИХ ПРИМЕНЕНИЕ ДЛЯ ПОЛУЧЕНИЯ АНТИТЕЛ С ПОВЫШЕННЫМИ АФФИННОСТЬЮ СВЯЗЫВАНИЯ Fc-РЕЦЕПТОРА И ЭФФЕКТОРНОЙ ФУНКЦИЕЙ |
WO2009028417A1 (ja) | 2007-08-24 | 2009-03-05 | Tokyo Institute Of Technology | 婦人科癌の検出方法 |
Also Published As
Publication number | Publication date |
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EP1162269A1 (en) | 2001-12-12 |
EP1162269A4 (en) | 2004-04-21 |
AU2691500A (en) | 2000-09-14 |
US7241605B1 (en) | 2007-07-10 |
JP2000245464A (ja) | 2000-09-12 |
CA2365019A1 (en) | 2000-08-31 |
JP4689047B2 (ja) | 2011-05-25 |
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