JPWO2002018602A1 - Novel Polypeptides - Google Patents

Abstract

(57) [Abstract] The present invention provides a novel polypeptide having β1,3-N-acetylglucosaminyltransferase activity, a glycosyltransferase containing the polypeptide, a method for producing glycosyltransferases and glycoconjugates using the glycosyltransferase, DNA encoding the polypeptide, a method for producing the polypeptide, an antibody against the polypeptide, and a diagnostic method and therapeutic drug for inflammation, cancer, or cancer metastasis using the DNA or the antibody. The present invention is useful for synthesizing useful glycosyltransferases and for diagnosing and treating inflammatory diseases, cancer, and cancer metastasis.

Description

Detailed Description of the InventionTechnical Field The present invention relates to lactosylceramide β1,3-N-acetylglucosaminyltransferase
activity and paragloboside β1,3-N-acetylglucosaminyltransferase activity
and a glycosyltransferase containing the polypeptide as an active ingredient.
a DNA encoding said polypeptide; a method for treating inflammation, cancer or cancer metastasis containing said DNA;
a recombinant DNA obtained by incorporating the DNA into a vector;
Transformant carrying the recombinant DNA, and production of the polypeptide using the transformant
a method for producing a sugar chain or a glycoconjugate using the polypeptide;
a method for producing the sugar chain or glycoconjugate obtained from DNA encoding the polypeptide;
A method for detecting inflammation, cancer or cancer metastasis using an oligonucleotide containing the polypeptide
an antibody that recognizes the antibody, an immunohistochemical staining method using the antibody, and an immunohistochemical staining method containing the antibody
staining agent, diagnostic agent for inflammatory disease, cancer or cancer metastasis, said polypeptide, said DNA, said
Recombinant vector, or a pharmaceutical containing said antibody, lactosylation of said polypeptide
Ceramide β1,3-N-acetylglucosaminyltransferase activity and paraglobulin
Screening of compounds that alter β1,3-N-acetylglucosaminyltransferase activity
screening method, a screening method for compounds that alter the expression of the gene,
The promoter DNA that controls the transcription of the gene, and the efficiency of transcription by the promoter DNA
screening methods for compounds that vary the
and non-human knockout animals in which the gene is deleted or mutated.
Regarding.Background technologyLactosylceramide β1,3-N-acetylglucosaminyltransferase is a
Galβ1-4Glc-ceramide (Galβ1-4Glc-ceramide)
It has the activity of transferring N-acetylglucosamine to inctoose residues via a β1,3 bond.
It is an enzyme that produces lactosylceramide (Galβ1-4Glc-ceramide).
, neolactoglycolipids, lactoglycolipids, ganglioglycolipids, globoglycolipids
, and isoglobo-series glycolipids are synthesized, but lactosylceramide β1,3-N
-Acetylglucosaminyltransferase synthesizes neolactoglycolipids and lactoglycolipids.
It is a key enzyme in the synthesis of lactosylceramide. When GM3 synthase acts on lactosylceramide, ganglioside GM3 (Neu
Acα2-3Galβ1-4Glc-ceramide) is synthesized.
When GM2 synthase acts on mide, asialoGM2 (GalNAcβ1-4Galβ
1-4Glc-ceramide) is synthesized. Many other compounds are synthesized from GM3 and asialoGM2.
Since many gangliosides are synthesized, GM3 synthase and GM2 synthase are
It can be said to be a key enzyme in the synthesis of ganglioglycolipids.
When tosylceramide α1,4-galactosyltransferase acts, Galα1-4Gal
β1-4Glc-ceramide is synthesized, followed by a series of globo-series glycolipids.
Lactosylceramide is converted to lactosylceramide α1,3-galactosyltransferase
When activated, Galα1-3Galβ1-4Glc-ceramide is synthesized, followed by
A series of isogloboglycolipids are synthesized. Therefore, lactosylceramide α1,4-galactosyltransferase and lactosylceramide
galactoside α1,3-galactosyltransferase is a globoglycolipid and isoglycolipid enzyme, respectively.
It is a key enzyme in the synthesis of globoglycolipids.
The synthesis of is controlled by the expression and expression level of the key enzymes mentioned above.
It is thought that neolacto-series glycolipids are Galβ1-4GlcNAcβ1-3Galβ1-4G
It is a glycolipid having an lc-ceramide skeleton, and lacto-series glycolipids are Galβ1-3G
It is a glycolipid having an lcNAcβ1-3Galβ1-4Glc-ceramide backbone.
Representative neolactoglycolipids include paragloboside (Galβ1-4Gl
cNAcβ1-3Galβ1-4Glc-ceramide), sialylparagloboside
(NeuAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Gl
c-ceramide), NeuAcα2-3Galβ1-4(Fucα1-3)Glc
NAcβ1-3Galβ1-4Glc-ceramide, etc. Typical lacto-type
Glycolipids include Galβ1-3GlcNAcβ1-3Galβ1-4Glc-
Ceramide, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1
-4Glc-ceramide, NeuAcα2-3Galβ1-3(Fucα1-4)
GlcNAcβ1-3Galβ1-4Glc-ceramide. In many human cancers (especially colon cancer or stomach cancer), fucose and sialic acid
It was found that lacto- or neolacto-type glycolipids were accumulated in large amounts.
[Annu. Rev. Immunol.,2, 103 (1984
), Chem. Phys. Lipids,42, 209 (1986)]. colorectal cancer
Glycosyltransferase activity in tissues and surrounding normal tissues, and in various colon cancer cell lines
As a result of measuring lactosylceramide β1,
It was found that the activity of 3-N-acetylglucosaminyltransferase was increased [
J. Biol. Chem. ，262, 15649 (1987)]. This result is
Increased lacto- or neolacto-series glycolipids in colorectal cancer may be due to lactosylceramide
caused by increased activity of mido-β1,3-N-acetylglucosaminyltransferase
This suggests that the human promyelocytic cell line HL-60 is
Treatment with tylsulfoxide or retinoic acid resulted in granulocytic cells
On the other hand, HL-60 cells were differentiated by phorbol-12-myristate-13-acetate.
When treated with phorbol esters such as paramethicone (PMA), they differentiate into monocyte-macrophages.
During differentiation into granulocytic cells, neolactoglycolipids (paragloboside)
and sialylparagloboside) increased and ganglioside GM3 decreased,
During differentiation into monocyte-macrophages, ganglioside GM3 increases and neola
In addition, neolactoglycolipids were added to HL-60 and cultured.
When cultured, they differentiate into granulocyte cells, and when ganglioside GM3 is added to HL-60,
These results suggest that the specific glycolipids
This indicates that the expression of HL-6 is important for differentiation induction and determining the direction of differentiation.
When 0 was treated with retinoic acid, GM3 synthase activity was not changed, but lactosylation
Ceramide β1,3-N-acetylglucosaminyltransferase activity increases [J. B
iol. Chem. ，267, 23507 (1992)]. Therefore, retinoin
In acid-treated HL-60, lactosylceramide β1,3-N-acetyl
Increased glucosaminyltransferase activity leads to an increase in neolactoglycolipids.
It causes a decrease in ganglioside GM3, resulting in granulocytic cells.
On the other hand, when HL-60 cells are treated with PMA, the GM3 synthase is activated.
Increased enzyme activity and lactosylceramide β1,3-N-acetylglucosaminyl transferase
The enzyme activity is reduced [J. Biol. Chem.,267, 23507 (199
2). Therefore, in HL-60 cells treated with PMA, there was an increase in GM3 synthase activity.
and decreased lactosylceramide β1,3-N-acetylglucosaminyltransferase activity
This causes an increase in ganglioside GM3 and a decrease in neolactoglycolipids.
This is thought to result in differentiation into monocyte-macrophages.
Tosylceramide β1,3-N-acetylglucosaminyltransferase and GM3 synthase
It is thought to play an important role in inducing differentiation of promyelocytic cells and determining their differentiation direction.
It has been found that leukocytes express different glycolipids depending on their type and differentiation stage.
For example, mature myeloid cells express neutral neolactoglycolipids.
It expresses only [Mol. Cell. Biochem.,47, 81 (198
2), J. Biol. Chem. ，260, 1067 (1985)].
Mature lymphocytes express only globoglycolipids [Mol. Cell
．． Biochem. ，47, 81 (1982)].
Therefore, the difference between the above glycolipids is lactosylceramide β1,3-N-acetylglucosamine.
It has been suggested that the difference in transaminase activity is the cause.
In myeloid cell lines such as G-1 and HL-60, lactosylceramide β1,3-N
-Acetylglucosaminyltransferase activity was detected, whereas Reh, CCRF
Lymphoid cells such as CEM, MOLT-4, Ramos, RPMI8226
It has been revealed that this enzyme activity is not detected in the strain [Archves
of Biochemistry and Biophysics,303, 1
25 (1993)]. Glycolipids having glucuronic acid 3-sulfate at the non-reducing end of the sugar chain (e.g., SO₄3
GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-
Ceramides are known to be expressed at specific times and in specific locations during the differentiation of the nervous system.
These glycolipids are known to play important roles in the mutual recognition of cells in the nervous system and in nerve migration.
It has been suggested that it is involved in the synthesis of ATP [J. Biol. Chem.,2
73Glucuronic acid 3-sulfate-containing sugars in nerve cells
Lipids (SO₄3GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ
The expression of lactosylceramide β1,3-N-acetyl
Since it is regulated by glucosaminyltransferase, lactosylceramide
Expression of β1,3-N-acetylglucosaminyltransferase enhances neuronal recognition.
It is thought that recognition and migration are controlled by this [J. Biol.
Chem.,273Glucuronic acid 3-sulfate is a compound found in human
It is also recognized by the monoclonal antibody HNK-1 against a marker of NK cells.
Therefore, it is also called the HNK-1 epitope.
Acid-containing glycolipids are also thought to play an important role in NK cell function.
Sugar chains with a GlcNAcβ1-3Gal structure are either neolacto- or lacto-based.
It is present in the sugar chains of glycolipids, as well as in the N-glycosidic sugar chains and O-glycosidic sugar chains of glycoproteins.
It also occurs in glycosidic sugar chains and oligosaccharides. For example, GlcNA
Oligosaccharides having a cβ1-3Gal structure include lactose, which is present in human milk.
-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4
Glc) and lacto-N-tetraose (Galβ1-3GlcNAcβ1-3G
alβ1-4Glc), or various oligosaccharides with these as the backbone.
[Acta Paediatrica,82, 903 (1993)]. Glc
The NAcβ1-3Gal structure is a component of poly-N-acetyllactosamine sugar chains.
Poly-N-acetyllactosamine sugar chains are also
A structure in which α-aspartic acid is repeatedly linked with β1,3 bonds [(Galβ1-4GlcNAc
β1-3)_n; n is 2 or more], and N-glycosidic bonds of glycoproteins
It is found in glycosyltransferases and O-glycosidic glycosyltransferases, as well as in glycosyltransferases of glycolipids and oligosaccharides.
It is also found in sugars. Lactosylceramide β1,3-N-acetylglucosamine
The transferase can also transfer substrates other than lactosylceramide, such as paragloboside and glycoproteins.
N-glycosidic sugar chains and O-glycosidic sugar chains of proteins, or oligosaccharides
It is not clear whether sugars are used as substrates. Lactosylceramide β1,3-N-acetylglucosaminyltransferase activity is
So far, colon tissue, colon cancer tissue, colon cancer cell lines (Colo205, SW403, etc.)
), and myeloid cell lines (K-562, KG-1, HL-60).
However, lactosylceramide β1,3-N-acetylglucosaminyltransferase is highly purified.
There have been no reports of its purification to such a high degree [J. Biol. Chem.,262, 15649 (
1987), Archves of Biochemistry and Bi
ophics,260, 461 (1988), Carbohydrate
Research,209, 261 (1991), Archves of Bi
ochemistry and biophysics,303, 125 (19
93)]. On the other hand, N-acetylglucosamine is linked to the galactose residue at the non-reducing end of the sugar chain via a β1,3 bond.
An enzyme with the activity of transferring cetylglucosamine (hereinafter referred to as Galβ1,3-N-acetylglucosamine)
Regarding cetylglucosaminyltransferase, there have been no reports of partial purification.
However, it is unclear whether these enzymes use lactosylceramide as a substrate.
[J. Biol. Chem.,268, 27118 (1993),
J. Biol. Chem. ，267, 2994 (1992), J. Biol. C
hem.,263, 12461 (1988), Jpn. J. Med. Sci. B
iol.,42, 77 (1989)]. To date, two types of Galβ1,3-N-acetamide genes have been cloned.
The gene for acetylglucosaminyltransferase has been cloned [Proc. Natl.
l. Acad. Sci. USA,94, 14294-14299 (1997),
Proc. Natl. Acad. Sci. USA,96, 406-411 (19
99)]. One of these, β3GnT, isin vitroIn paragraph
It has been shown that the substrate is loboside, but the substrate is lactosylceramide.
In this case, the activity is weak [Glycobiology,9, 1123 (1999)].
In addition, β3GnT uses lactosylceramide and paragloboside as substrates within cells.
It is not clear whether another Galβ1,3-N-
It is unknown whether cetylglucosaminyltransferase exists. There are a great number of glycans with the GlcNAcβ1-3Gal structure.
Therefore, Galβ1,3-N-acetylglucosaminyltransferase has acceptor substrate specificity
There may be multiple enzymes with different expression levels and tissues, each with a different function.
Therefore, unlike the two enzymes that have been cloned so far,
Different Galβ1,3-N-acetylglucosaminyltransferases were cloned and
By examining the substrate specificity, lactosylceramide β1,3-N-acetyl
It is believed that glucosaminyltransferase can be identified. As mentioned above, lacto-N-neotetraose (Galβ1) is found in human milk.
-4GlcNAcβ1-3Galβ1-4Glc) and lacto-N-tetraose
(Galβ1-3GlcNAcβ1-3Galβ1-4Glc), or
It is known that there are various oligosaccharides having a structure with these as the core [A
cta Paediatrica,82, 903 (1993)]. The oligosaccharide is
They all have a GlcNAcβ1-3Gal structure.
These also include oligosaccharides having poly-N-acetyllactosamine sugar chains.
The oligosaccharides in this product have the function of preventing infants from being infected by viruses and microorganisms and of neutralizing toxins.
It is also believed to have a harmonizing effect.
On the other hand, it also has the activity of promoting the proliferation of bacteria.
The types of sugars present in human milk are few, with the majority being lactose.
There are almost no oligosaccharides in human milk. The above oligosaccharides contained in human milk, or milk containing them, are efficiently
If it can be produced effectively, it will be very useful industrially.
Galβ1,3-N-acetylglucosaminyl transfer involved in the synthesis of the above oligosaccharides
If the gene for the enzyme can be obtained, it is thought that it can be used for the efficient synthesis of the above oligosaccharides.
However, the enzyme is not yet known. Among glycans with the GlcNAcβ1-3Gal structure, poly-N-acetyl
Lulactosamine glycans are involved in many functional glycans (selectin ligand glycans, microbial and
It is the backbone glycan of various proteins (such as viral receptor glycans, SSEA-1 glycans, and cancer-related glycans).
They are deeply involved in embryonic development, cell differentiation, and diseases such as inflammation and cancer.
Poly-N-acetyllactosamine sugar chains are also important for stabilizing glycoproteins.
It plays a vital role in the synthesis of poly-N-acetyllactosamine sugar chains, which function in each of these areas.
The Galβ1,3-N-acetylglucosaminyltransferases involved may be different.
Therefore, it is different from the two enzymes cloned so far.
N-acetylglucosaminyltransferase may be present.
In addition to lactosylceramide, β1,3-N-acetylglucosaminyltransferase
, having Galβ1-4Glc or Galβ1-4GlcNAc at the non-reducing end
N-acetyllactosamine is also transferred to sugar chains (e.g., paragloboside) that are involved in the synthesis of poly(N-acetyllactosamine).
It may be involved in the synthesis of poly-N-acetyllactosamine glycans. Below, we will describe the synthesis, functions, and applications of poly-N-acetyllactosamine glycans.
Poly-N-acetyllactosamine sugar chains consist of GlcNAcβ1,4-galactosamine
glycosyltransferase (β1 catalyzes the binding of N-acetylglucosamine residues at the non-reducing end of sugar chains)
,4-galactose transfer enzyme) and Galβ1,3-N-
It is synthesized by alternating acetylglucosaminyltransferase activity.
Regarding NAcβ1,4-galactosyltransferase, four types of enzymes (
β4Gal-T1, β4Gal-T2, β4Gal-T3, β4Gal-T4)
The genes for these enzymes have been cloned and the acceptor substrate specificity of each enzyme has been analyzed.
[J. Biol. Chem.272, 31979-31991 (1997), J
．． Biol. Chem.273, 29331-29340 (1997)]. Fucose is present on the linear or branched poly-N-acetyllactosamine sugar chain.
, sialic acid, N-acetylgalactosamine, sugars such as galactose, or sulfate
Various sugar chains (functional sugar chains, blood sugar chains) are added to the sugar chains in a cell-specific or time-specific manner.
Liquid-type glycans, cancer-related glycans, etc.) are formed [Kiwata Akira, Hakomori Senichiro, Nagai Katsutaka,
Glycobiology Series, Kodansha, (1993). On granulocytes, monocytes, and activated T cells, sialyl Lewis x sugars are attached to the ends of the sugar chains.
chain [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc]
It is known that poly-N-acetyllactosamine sugar chains exist,
These sugar chains act as ligands for the adhesion molecules E-selectin and P-selectin.
It is believed that these proteins function as a mediator between the inflammatory process and the leukocytes, and are involved in the accumulation of these leukocytes at the site of inflammation.
[Kiwata Akira, Hakomori Senichiro, and Nagai Katsutaka, eds., Glycobiology Series,
Kodansha, (1993)]. In addition, sialyl Lewis x sugar chains and sialyl Lewis x sugar chains are also present at the terminals of cancer cells, such as those in colon cancer.
Is a sugar chain [NeuAcα2-3Galβ1-3(Fucα1-4)GlcNA
It is known that poly-N-acetyllactosamine sugar chains having the structure
These sugar chains also function as ligands for E-selectin and P-selectin.
It has been suggested that it is involved in cancer metastasis.
[Edited by Katsutaka, Glycobiology Series, Kodansha, (1993)]. The structure of poly-N-acetyllactosamine sugar chains is known to play a key role in embryonic development, cell differentiation, or cell differentiation.
It is known that the cytoplasmic endothelial cells change during the process of canceration.
Edited by Glycobiology Series, Kodansha, (1993).
Linear poly-N-acetyllactosamine sugar chains are expressed in the erythrocytes of infants.
In contrast, branched poly-N-acetyllactosamine sugar chains are expressed in adult erythrocytes.
[Kiwata Akira, Hakomori Senichiro, and Nagai Katsutaka, eds., Glycobiology Series "Sugar Chain"
The diverse world of these poly-N-acetylglucosamines on red blood cells.
The ABO blood group antigens are expressed at the end of the lactosamine sugar chain.
Blood group antigens are expressed at each end of the poly-N-acetyllactosamine sugar chain.
When this happens, it becomes a multivalent antigen, and the binding ability of antibodies to blood group sugar chains is different from that of linear antigens.
Compared to 10³During early mouse embryonic development, a series of carbohydrate antigens are regularly expressed.
It is known that SSEA-1 (stage specific embryo
The ryonic antigen-1 antigen is poly-N-acetyllactosamine
Lewis x sugar chains [Galβ1-4(Fucα1-3)GlcN
Ac], but expression of this antigen begins at the 8-cell stage and peaks in the morula,
It gradually disappears after the blastocyst stage (Kiwata Akira, Hakomori Senichiro, Nagai Katsutaka, eds., Glico
Biology Series "Glycobiology of Cellular Society", Kodansha, 1993
The morula stage is the embryonic stage, which has previously undergone simple numerical growth through cell division.
During the transitional period when cells move into the blastocyst stage, which is the stage where they first develop a differentiated "morphology"
The cells of the morula are packed tightly together just before forming the blastocyst.
It causes cell compaction. It has SSEA-1 antigen.
Addition of oligosaccharides inhibits this cell compaction and subsequent normal development.
Harmful [J. Exp. Med.,160, 1591 (1984)].
Mouse teratocarcinoma cell adhesion is inhibited by anti-SSEA-1 antibodies
It is also known that [Kiwata Akira, Hakomori Senichiro, and Nagai Katsutaka (eds.), Glycobiology Series
[Rees, "Glycobiology of Cellular Society," Kodansha, 1993].
The SSEA-1 antigen acts as an adhesion molecule or a sugar chain signal, and is involved in the development of early embryonic development.
This indicates that cancer cells contain significantly more poly-N-acetyllactosamine than corresponding normal cells.
It is known that it expresses a large amount of sugar chains [J. Biol. Chem.,259,
10834 (1984), J. Biol. Chem. ，261, 10772 (1
986), J. Biol. Chem. ，266, 1772 (1991), J. B
iol. Chem. ，267, 5700 (1992)].
When the -ras proto-oncogene is expressed, the molecules of N-linked glycans on the cell surface are
The amount of N-linked glycans increases, and the cells acquire invasive ability.
As the amount of N-acetyllactosamine sugar chains increases, the amount of poly-N-acetyllactosamine sugar chains also increases.
β1,4-galactosyltransferase and β1,3-N-galactosamine sugar chain synthesis
It is known that the activity of acetylglucosaminyltransferase is increased [J
．． Biol. Chem. ，266, 21674 (1991)]. Galectins are a group of lectins that have affinity for β-galactosides and act on cells.
It is involved in adhesion and signal transduction, and its association with diseases such as cancer has also been suggested.
s in Glycoscience and Glycotechnology
y,9, 9 (1997)]. Ten types have been known to date in mammals.
Among these, galectin-1 and -3 are linear poly-N-acetyllactosaccharides.
It is known that the saccharide chains bind with high affinity to the saccharide chains.
Certain glycoproteins are putative ligands for these galectins.
[Trends in Glycoscience and Glycotec
phology,9, 9 (1997), Trends in Glycosci.
ence and Glycotechnology,9, 47 (1997)]
Poly-N-acetyllactosamine sugar chains with sialic acid attached to the terminal end are
It acts as a receptor for plasma and microorganisms [Acta Paediatrica,8
2, 903 (1993)]. In this way, poly-N-acetyllactosamine sugar chains can be used to form many functional sugar chains (saccharin-containing sugar chains).
Lectin ligand glycans, microbial and viral receptor glycans, SSEA-1 glycans, cancer
and blood group-related glycans) and blood group glycans, and efficiently presents these glycans.
Poly-N-acetyllactosamine glycans with sialyl Lewis x glycans play an important role in
As an antagonist of cutin, it is a drug that has anti-inflammatory effects or suppresses cancer metastasis.
It is expected to be a drug. It has polyvalent (four) sialyl Lewis x sugars on a poly-N-acetyllactosamine sugar chain.
In oligosaccharides with a chain (tetrasaccharide) bonded to them, the sialyl Lewis x oligosaccharide is not multivalent.
Compared to tetrasaccharides, it acts as a selectin antagonist at a concentration of 1/100 or less.
It is known that it exhibits activity as a steroid hormone [J. Exp. Med.,182, 113
3 (1995), Glycobiology,6, 65 (1996), Glyc.
biology,7, 453 (1997), Eur. J. Immunol. ，
27, 1360 (1997)]. The poly-N-acetyllactosaccharides of these oligosaccharides
For the synthesis of the glycosaminoglycan moiety, partially purified β1,3-N-acetylglucosamine was used.
However, the supply of this enzyme was rate-limiting, and poly-N-acetyltransferase was used.
It is difficult to synthesize lactosamine sugar chains in large quantities [Glycobiology,
7, 453 (1997)]. On the other hand, poly-N-acetyllactosamine sugar chains can also be synthesized by chemical synthesis.
However, the synthesis requires very complicated steps [Tetrahydro
n Letter,24, 5223 (1997)]. Based on the above, an efficient method for synthesizing poly-N-acetyllactosamine sugar chains is needed.
Two Galβ1,3-N-acetyltransferases have been cloned so far.
Alternatively, glucosaminyltransferase or the enzyme gene can be used, but depending on the purpose,
The substrate specificity and function of other Galβ1,3-N-acetylglucosamine transferases are different.
Transferases (e.g., lactosylceramide β1,3-N-acetylglucosaminyltransferases)
It is thought that in some cases it may be more efficient to use a poly-N-acetyllactosamine sugar chain or the enzyme gene. Poly-N-acetyllactosamine sugar chains are also important for stabilizing glycoproteins.
Ru. lysosome associated membrane glyco
protein-1 (lamp-1) and lysosome associa
ted membrane glycoprotein-2 (lamp-2)
, a glycoprotein present in lysosomes (some are also present on the cell surface),
It almost completely covers the inner surface of the endothelial membrane.
2 contains many sugar chains (some of which contain poly-N-acetyllactosamine sugar chains).
lamp-1 and lamp-2 are hydrolases in the lysosome.
The human promyelocytic cell line HL-60 was used to prevent the degradation of the cells.
When treated with dimethyl sulfoxide
During this differentiation process, lamp-1 and lam
The number of poly-N-acetyllactosamine sugar chains added to p-2 increases, and
The metabolic rate (decomposition rate) of both lamp-1 and lamp-2 is reduced.
It is known [J. Biol. Chem.,265, 20476 (1990)]
. Examples of increased poly-N-acetyllactosamine sugar chain synthesis ability include the following:
Examples include treatment of F9 cells with retinoic acid and treatment of Swiss3T3 cells with TGF-
When treated with β, poly-N-acetylglucosamine was attached to the sugar chains of membrane-bound glycoproteins of cells.
It has been shown that a ctosamine sugar chain is added to the glycan [J. Biol. Chem.,2
68, 1242 (1993), Biochim. Biophys. Acta. ，
1221, 330 (1994)]. When the N-ras proto-oncogene was expressed in NIH3T3 cells, poly-N
-β1,4-galactosyltransferase involved in the synthesis of acetyllactosamine sugar chains
and β1,3-N-acetylglucosaminyltransferase activity increased, and membrane protein
The amount of poly-N-acetyllactosamine sugar chains in the N-linked sugar chains of
It is known [J. Biol. Chem.,266, 21674 (1991)]
The core 2 β1,6-N-acetylglucosaminyltransferase gene was expressed in the T cell line EL-4.
When the gene is expressed, the cell surface membrane proteins CD43, CD45, and
The molecular weight of CD44 increases [J. Biol. Chem.,271, 1873
2 (1996)]. This is a core 2 β1,6-N-acetylglucosaminyltransferase.
The sugar chains synthesized by the enzyme are involved in the synthesis of poly-N-acetyllactosamine sugar chains.
This is thought to be because it is a good substrate for β1,3-N-acetylglucosaminyltransferase, which
In addition, when HL-60 cells are cultured at 27°C, lamp-1 or lamp-
It is known that the amount of poly-N-acetyllactosamine sugar chains added to 2 increases.
[J. Biol. Chem.,266, 23185 (1991)]. However, host cells suitable for the production of recombinant glycoproteins (e.g., Namalwa
cells, Namalwa KJM-1 cells, CHO cells, etc.)
- Efficient production of recombinant glycoproteins with acetyllactosamine sugar chains
Therefore, it has not been reported that poly-N-acetyllactide
Efficient method for producing recombinant glycoproteins with tosamine sugar chains - Patent Application 20070122633
The development of Galβ1,3-N-acetylglucosamine is an important industrial challenge. The two Galβ1,3-N-acetylglucosamines that have been cloned so far are
Although it is possible to use a transaminase gene, it is necessary to select a gene with a specific substrate and function depending on the purpose.
A different Galβ1,3-N-acetylglucosaminyltransferase gene (e.g.,
, lactosylceramide β1,3-N-acetylglucosaminyltransferase)
It may be more efficient.Disclosure of the Invention The object of the present invention is to provide a method for the preparation of a compound having β1,3-N-acetylglucosaminyltransferase activity.
Using novel polypeptides, synthesis of useful sugar chains, anti-inflammatory, anti-infectious, anti-cancer metastasis, etc.
Improvement methods for pharmaceuticals, dairy products, and protein stabilization, as well as the treatment of inflammatory diseases and cancer
The present invention aims to provide a method for diagnosing the type and degree of malignancy of a tumor. The present invention relates to the following (1) to (79): (1) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. (2) A polypeptide consisting of the amino acids 39 to 378 of the amino acid sequence represented by SEQ ID NO: 1.
(3) A polypeptide comprising the amino acid sequence of the polypeptide according to (1) or (2).
and the amino acid sequence is one in which one or more amino acids are deleted, substituted, or added,
and a polypeptide having β1,3-N-acetylglucosaminyltransferase activity. (4) A polypeptide having a sequence identical to that of the amino acid sequence of the polypeptide according to (1) or (2) above, but with a sequence similar ...).
% or more homology to the amino acid sequence of
A polypeptide having cosaminyltransferase activity. (5) A polypeptide having β1,3-N-acetylglucosaminyltransferase activity that inhibits non-reducing glycans.
N-acetylglucosamine is attached to the terminal galactose residue via a β1,3 bond.
(6) The polypeptide according to any one of (3) and (4), wherein the β1,3-N-acetylglucosaminyltransferase activity is i) galactosidase activity.
N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1
-3GlcNAc or lactose (Galβ1-4Glc), ii) galactose
lactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose
Oligosaccharides having a galactose structure at the non-reducing end, and iii) galactose, N-acetylglucose
Non-reducing lactosamine, Galβ1-3GlcNAc or lactose structure
The galactose present at the non-reducing end of the sugar chain of the acceptor substrate selected from the terminal glycoconjugates is
It has the activity of transferring N-acetylglucosamine to the β1,3 bond of the β1,3 bond.
(7) The polypeptide according to any one of (3) to (5), wherein the polypeptide is selected from the group consisting of galactose, N-acetyllactosamine, and Galβ1-3GlcN.
A complex carbohydrate having a lactose structure at the non-reducing end is lactosylceramic acid.
ceramide (Galβ1-4Glc-ceramide) or paragloboside (Galβ1-4
GlcNAcβ1-3Galβ1-4Glc-ceramide)
(8) Complex carbohydrates include glycoproteins, glycolipids, proteoglycans, and glycopeptides.
, lipopolysaccharides, peptidoglycans, and glycosides with sugar chains attached to steroid compounds
(9) A method for treating a stomach ulcer, comprising administering to a patient a therapeutically effective amount of the polypeptide according to any one of (1) to (8) above, wherein the polypeptide is a complex carbohydrate selected from the group consisting of steroids, steroid hormone ...
(10) A glycosyltransferase encoding the polypeptide according to any one of (1) to (8).
DNA. (11) DNA having the base sequence represented by SEQ ID NO: 2. (12) The base sequence from bases 135 to 1268 of the base sequence represented by SEQ ID NO: 2.
(13) A DNA having the 249th to 1268th bases of the base sequence represented by SEQ ID NO: 2.
(14) A DNA having the base sequence of the DNA according to any one of (10) to (13).
It hybridizes under stringent conditions with DNA having a base sequence complementary to the sequence.
and a polypeptide having β1,3-N-acetylglucosaminyltransferase activity.
DNA encoding a peptide. (15) β1,3-N-acetylglucosaminyltransferase activity is a non-reducing enzyme of the glycan.
N-acetylglucosamine is attached to the galactose residue at the original terminal via a β1,3 bond.
(16) The DNA according to (14), wherein the β1,3-N-acetylglucosaminyltransferase activity is i) galactosidase activity.
N-acetyllactosamine (Galβ1-4GlcNAc), Galβ
1-3GlcNAc or lactose (Galβ1-4Glc), ii) galactose
lactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose
and ii) oligosaccharides having a galactose structure at the non-reducing end, and
Non-reducing lactosamine, Galβ1-3GlcNAc or lactose structure
The galactose present at the non-reducing end of the sugar chain of the acceptor substrate selected from the terminal glycoconjugates is
It has the activity of transferring N-acetylglucosamine to the β1,3 bond of the β1,3 bond.
(14) or (15). (17) The DNA according to (14) or (15), which is a galactose, N-acetyllactosamine, or Galβ1-3Glc
A complex carbohydrate having a NAc or lactose structure at the non-reducing end is called lactosylceramic acid.
(16) The DNA according to (16), wherein the complex carbohydrate is a glycoprotein, a glycolipid, a proteoglycan, a glycopeptide, or a paragloboside. (18) The DNA according to (16), wherein the complex carbohydrate is a glycoprotein, a glycolipid, a proteoglycan, a glycopeptide, or a glycopeptide.
Glycosides with sugar chains attached to glycosides, lipopolysaccharides, peptidoglycans, and steroid compounds
The DNA according to (16) or (17), which is a complex carbohydrate selected from the group consisting of nucleotides, nucleotides, and nucleotide sequences. (19) The DNA according to any one of (10) to (18),
(20) A DNA having a base sequence complementary to the DNA of any one of (10) to (18) incorporated into a vector.
(21) A recombinant vector obtained by incorporating a sequence homologous to the DNA described in any one of (10) to (18).
(22) A transformant having the recombinant vector according to (20) or (21).
Transformants. (23) Transformants consist of microorganisms, animal cells, plant cells, and insect cells.
The transformant according to (22), which is a transformant selected from the group consisting of: (24) The microorganism isEscherichiaIt is a microorganism belonging to the genus
(25) The transformant according to (3). (26) The animal cell is a mouse myeloma cell, a rat myeloma cell, or
Mouse hybridoma cells, CHO cells, BHK cells, African green monkey kidney
pancreatic cells, Namalwa cells, Namalwa KJM-1 cells, human fetal kidney
(23) The animal cell is selected from the group consisting of human leukemia cells and human leukemia cells.
Transformants of the above plants. (26) Plant cells are transformed into tobacco, potato, tomato, carrot, soybean, and
Rana, alfalfa, rice, wheat, barley, rye, corn, or flax
(23) The transformant according to (23), which is a plant cell selected from the group consisting of plant cells of
. (27) Insect cells,Spodoptera frugiperdaovaries
cell,Trichoplusia nifrom ovarian cells of silkworms and silkworms
(23) The transformant according to (23), which is an insect cell selected from the group consisting of: (28) The transformant according to (23), which is a non-human transgenic animal or a transgenic
(29) The transformant according to (22), which is a nicked plant. (29) The transformant according to (22) to (27) is cultured in a medium, and the culture is
(1) A culture of the polypeptide according to any one of (1) to (8)
(30) A method for producing the polypeptide, comprising raising the non-human transgenic animal according to (28) and collecting the polypeptide from (1) to (30).
(8) The polypeptide according to any one of (8) above is produced and accumulated in the animal,
and recovering the polypeptide from the culture medium.
(31) The method according to (30), wherein the accumulation is in animal milk.
(32) A method for producing a transgenic plant according to (28) above, comprising cultivating the transgenic plant according to (1) to (8).
The polypeptide according to any one of claims 1 to 4 is produced and accumulated in the plant, and
(33) A method for producing the polypeptide, comprising: using the DNA according to any one of (10) to (18),in
vitroThe transcription and translation system in
(34) A method for producing a polypeptide, comprising synthesizing the polypeptide using the glycosyltransferase according to (9) as an enzyme source, and synthesizing the polypeptide from (a) the enzyme source, (b) i) lactosylceramide (Galβ1-4Glc-ceramide) or paraglycosylceramide.
Globoside (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-sera
ii) galactose, N-acetyllactosamine (Galβ1-4Gl
cNAc), Galβ1-3GlcNAc or lactose (Galβ1-4G
lc), iii) galactose, N-acetyllactosamine (Galβ1-4G
lcNAc), Galβ1-3GlcNAc or lactose (Galβ1-4
Glc) oligosaccharides having a structure at the non-reducing end, and iv) galactose, N-
acetyllactosamine, Galβ1-3GlcNAc or lactose structure
(c) an acceptor substrate selected from complex carbohydrates having a reducing end, and (d) uridine-5'-diphosphate N-acetylglucosamine (UDP-GlcN)
Ac) is present in an aqueous medium, and the galactose acceptor substrate is added to the aqueous medium.
Sugar chains or complex sugars in which N-acetylglucosamine is attached to the residue via a β1,3 bond
and collecting the sugar chain or glycoconjugate from the aqueous medium.
(35) A method for producing the sugar chain or glycoconjugate, characterized by:
The provided glycan or glycoconjugate is used as an acceptor substrate, and (a) the acceptor substrate, (b) GlcNAcβ1,4-galactosyltransferase, and (c) uridine-5'-diphosphate galactose (UDP-Gal) are reacted in an aqueous medium.
and in the aqueous medium, N-acetylglucosamine at the non-reducing end of the acceptor substrate is introduced.
A reaction product in which galactose is attached to the samine residue via a β1,4 bond is generated and accumulated.
and collecting the sugar chain or glycoconjugate to which galactose has been added from the aqueous medium.
and (c) producing the sugar chain or glycoconjugate to which galactose has been added, the sugar chain or glycoconjugate being characterized by:
(36) A method for producing a glycosyltransferase comprising the steps of: (a) the enzyme source; (b) GlcNAcβ1,4-galactosyltransferase; and (c) i) lactosylceramide (Galβ1-4Glc-ceramide) or paraglycosylceramide (PGA).
Globoside (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-sera
ii) galactose, N-acetyllactosamine (Galβ1-4Gl
cNAc), Galβ1-3GlcNAc or lactose (Galβ1-4G
lc), iii) galactose, N-acetyllactosamine (Galβ1-4G
lcNAc), Galβ1-3GlcNAc or lactose (Galβ1-4
Glc) oligosaccharides having a structure at the non-reducing end, iv) galactose, N-acetyl
Lactosamine, Galβ1-3GlcNAc or lactose structure
and v) a complex carbohydrate having a terminal end, and a compound obtained by the method described in (34) or (35).
(d) an acceptor substrate selected from the group consisting of glycans or glycoconjugates that can be synthesized by uridine-5'-diphosphate N-acetyllactosamine (UDP-GlcN);
Ac), and (e) uridine-5'-diphosphate galactose (UDP-Gal) in an aqueous medium.
and in the aqueous medium, a poly-N-acetyl group is attached to the non-reducing end of the acceptor substrate.
A reaction product having a lactosamine sugar chain attached thereto is produced and accumulated, and the reaction product is extracted from the aqueous medium.
A sugar chain or a glycoconjugate having a poly-N-acetyllactosamine sugar chain attached thereto is collected.
The sugar chain having the poly-N-acetyllactosamine sugar chain attached thereto is characterized in that
(37) A method for producing a carbohydrate or complex carbohydrate, comprising: using the transformant according to any one of (22) to (27),
lcNAcβ1-3Galβ1-4Glc-ceramide, lacto-series glycolipid (Gal
β1-3GlcNAcβ1-3Galβ1-4Glc-ceramide as the backbone
glycolipids), neolacto-series glycolipids (Galβ1-4GlcNAcβ1-3Ga
GlcNAcβ1-
Sugars having a 3Gal structure, GlcNAcβ1-3Galβ1-4GlcNAc structure
sugars having a GlcNAcβ1-3Galβ1-3GlcNAc structure;
sugars, sugars having a GlcNAcβ1-3Galβ1-4Glc structure, (Galβ1
-4GlcNAcβ1-3)_nGalβ1-4GlcNAc structure and n is 1 or more
and (Galβ1-4GlcNAcβ1-3)_nGalβ1-4
a sugar chain comprising a sugar selected from the group consisting of sugars having a Glc structure and n being 1 or more;
Alternatively, a complex carbohydrate containing the sugar chain is produced and accumulated, and the sugar chain or
(38) A method for producing the sugar chain or glycoconjugate, comprising collecting the glycoconjugate. (39) A method for producing the sugar chain or glycoconjugate, comprising collecting the glycoconjugate from the non-human transgenic animal or transgenic mammal according to (28).
GlcNAcβ1-3Galβ1-4Glc-ceramide,
Glycolipids of the methicone series (Galβ1-3GlcNAcβ1-3Galβ1-4Glc-
glycolipids with a tetradecylamide backbone), neolacto-series glycolipids (Galβ1-4Gl
Glycolipids with cNAcβ1-3Galβ1-4Glc-ceramide as the backbone
, sugars having a GlcNAcβ1-3Gal structure, GlcNAcβ1-3Galβ
Sugars having a 1-4GlcNAc structure, GlcNAcβ1-3Galβ1-3Gl
sugars having a cNAc structure, sugars having a GlcNAcβ1-3Galβ1-4Glc structure
Sugars that are (Galβ1-4GlcNAcβ1-3)_nGalβ1-4GlcNA
Sugars having a c structure and n being 1 or greater, and (Galβ1-4GlcNAcβ1-
3)_nGalβ1-4Glc structure and n is 1 or more.
and producing and accumulating a sugar chain consisting of a sugar selected from the group consisting of sugars or a glycoconjugate containing the sugar chain,
The sugar chain or the complex carbohydrate is collected from the body.
Method for producing carbohydrates. (39) A method for producing a glycoconjugate comprising the steps of: glycoproteins, glycolipids, proteoglycans, and glycopeptides.
Glycosides with sugar chains attached to glycosides, lipopolysaccharides, peptidoglycans, and steroid compounds
(34) The method according to any one of (34) to (38), wherein the complex carbohydrate is selected from the group consisting of lactic acid bacteria, ...
(40) The method according to (38), wherein the accumulation is in animal milk.
(41) A method for producing a DNA comprising the base sequence of any one of (10) to (19).
an oligonucleotide having the same sequence as the consecutive 5 to 120 bases of the sequence;
(42) An oligonucleotide that is a derivative of a oligonucleotide. (43) The DNA according to any one of (10) to (19), a portion of the DNA
(41) A hybridization reaction using the fragment or the oligonucleotide according to (41)
A gene encoding the polypeptide according to any one of (1) to (8) is produced by a gene transcription method.
(43) A method for quantifying the expression level of a gene. (44) A method for quantifying the expression level of a gene by polymerase chain reaction using the oligonucleotide according to (41).
The polypeptide according to any one of (1) to (8) is obtained by the ion reaction method.
(44) A method for quantifying the expression level of a gene encoding a marker. (44) A method for quantifying the expression level of a gene encoding a marker, using the method according to (42) or (43).
(45) A method for detecting a mutation in a DNA according to any one of (10) to (19), a portion of the DNA
(41) A method for treating inflammation, cancer, or
A cancer metastasis detection agent. (46) A gene encoding the polypeptide according to any one of (1) to (8).
A method for diagnosing a functional abnormality of a gene by detecting a mutation in the gene. (47) A DNA according to any one of (10) to (19), a portion of the DNA
(41) A hybridization reaction using the fragment or the oligonucleotide according to (41)
A gene encoding the polypeptide according to any one of (1) to (8) is produced by a gene transcription method.
(48) A method for detecting a mutation in a gene. (49) A method for detecting a mutation in a gene using a polymerase chain reaction (PCR) method using the oligonucleotide according to (41).
The polypeptide according to any one of (1) to (8) is obtained by the ion reaction method.
(49) A method for detecting a mutation in a gene encoding a gene encoding a nucleotide sequence, comprising: (1) detecting a mutation in a gene encoding a nucleotide sequence according to any one of (1) to (8) using the oligonucleotide according to (41).
(50) A method for suppressing the transcription of a gene encoding a polypeptide or the translation of mRNA. (51) An antibody that recognizes the polypeptide according to any one of (1) to (8).
(51) A method for detecting a human leukemia virus according to any one of (1) to (8), wherein the antibody according to (50) is used.
(52) A method for immunologically detecting the polypeptide described in any one of (1) to (8) using the antibody described in (50).
(53) An immunohistochemical staining method for detecting a polypeptide comprising the antibody according to (50). (54) A pharmaceutical composition comprising the polypeptide according to any one of (1) to (8).
Medicine. (55) A medicine for the treatment, prevention and/or treatment of inflammatory diseases, cancer or cancer metastasis.
The pharmaceutical according to (54), which is a pharmaceutical for diagnosis. (56) The DNA according to any one of (10) to (19), a portion of the DNA
(57) A pharmaceutical comprising the fragment or the oligonucleotide according to (41). (57) A pharmaceutical comprising the fragment or the oligonucleotide according to (41), for use in the treatment, prevention, and/or prophylaxis of inflammatory diseases, cancer, or cancer metastasis.
The pharmaceutical according to (56), which is a pharmaceutical for diagnosis. (58) A pharmaceutical comprising the recombinant vector according to (20) or (21). (59) A pharmaceutical for treating, preventing, and/or treating an inflammatory disease, cancer, or cancer metastasis.
The pharmaceutical according to (58), which is a pharmaceutical for diagnosis. (60) A pharmaceutical comprising the antibody according to (50). (61) The pharmaceutical is a pharmaceutical for treating, preventing, and/or treating an inflammatory disease, cancer, or cancer metastasis.
The pharmaceutical according to (60), which is a pharmaceutical for diagnosis. (62) A pharmaceutical comprising a polypeptide according to any one of (1) to (8) and a test sample.
and contacting the test sample with the β1,3-N-acetylglucosamine contained in the polypeptide.
The polypeptide is characterized by measuring fluctuations in glucosaminyltransferase activity.
of compounds that alter the β1,3-N-acetylglucosaminyltransferase activity
Screening method. (63) β1,3-N-acetylglucosaminyltransferase activity is a key factor in determining whether or not a sugar chain is non-reduced.
N-acetylglucosamine is attached to the galactose residue at the original terminal via a β1,3 bond.
(64) The screening method according to (62), wherein the β1,3-N-acetylglucosaminyltransferase activity is an activity to transfer galactose.
N-acetyllactosamine (Galβ1-4GlcNAc), Galβ
1-3GlcNAc or lactose (Galβ1-4Glc), ii) galactose
lactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose
and iii) oligosaccharides having a galactose structure at the non-reducing end,
Cetyllactosamine, Galβ1-3GlcNAc or lactose structure
The glycan present at the non-reducing end of the sugar chain of the acceptor substrate selected from the complex carbohydrates having the original end
It has the activity of transferring N-acetylglucosamine to lactose residues via a β1,3 bond.
The screening method according to (62) or (63), wherein the galactose, N-acetyllactosamine, Galβ1-3Glc
A complex carbohydrate having a NAc or lactose structure at the non-reducing end is called lactosylceramic acid.
(64) The screening method according to (64), wherein the complex carbohydrate is a glycoprotein, a glycolipid, a proteoglycan, a glycopeptide, or a paragloboside. (66) The screening method according to (64), wherein the complex carbohydrate is a glycoprotein, a glycolipid, a proteoglycan, a glycopeptide, or a glycopeptide.
Glycosides with sugar chains attached to glycosides, lipopolysaccharides, peptidoglycans, and steroid compounds
(64) or (65), wherein the complex carbohydrate is selected from the human body.
(67) A method for producing a cell line expressing the polypeptide according to any one of (1) to (8).
The cells are contacted with a test sample, and an antibody that recognizes paragloboside or poly-N-
A compound selected from the group consisting of antibodies and lectins that recognize acetyllactosamine sugar chains.
At least one of paragloboside or poly-N-acetyllactosamine is used.
The expression of a gene encoding the polypeptide is characterized by quantifying the sugar chains.
(68) A method for screening for a compound that alters the expression level of a polypeptide according to any one of (1) to (8) above, comprising:
and quantifying the polypeptide using the antibody according to (50).
A screening of compounds that alter the expression of the gene encoding the polypeptide is performed.
(69) A method for cloning a gene encoding the polypeptide according to any one of (1) to (8).
Promoter DNA controls gene transcription. (70) Promoter DNA is involved in the transcription of genes in white blood cells, nerve cells, tracheal cells, and lung cells.
Cells, colon cells, placental cells, neuroblastoma cells, glioblastoma cells, colon
A cell selected from cancer cells, lung cancer cells, pancreatic cancer cells, gastric cancer cells, and leukemia cells.
(69) The promoter DNA according to (69), which is a functioning promoter. (71) The promoter DNA is a promoter derived from a human, rat, or mouse.
(72) The promoter DNA according to any one of (69) to (71).
and a plasmid containing a reporter gene linked downstream of the promoter DNA.
transforming animal cells with the smid, and contacting the transformant with a test sample;
The method for detecting a reporter gene in a promoter, comprising quantifying a translation product of the reporter gene.
A method for screening compounds that alter the efficiency of transcription by chloramphenicol acetyltransferase. (73) A method for screening compounds that alter the efficiency of transcription by chloramphenicol acetyltransferase.
galactosidase gene, β-galactosidase gene, β-lactamase gene,
Selected from the ferase gene and green fluorescent protein gene
(72) The screening method according to (72), wherein the gene is a gene that can be detected. (74) The screening method according to any one of (62) to (68), (72), and (73).
(75) A compound obtained by the screening method described above. (76) A compound obtained by screening a polypeptide according to any one of (1) to (8) containing a deletion or deletion of DNA encoding the polypeptide.
(76) The knockout animal according to (75), wherein the non-human knockout animal is a mouse.
(77) A DNA according to any one of (10) to (18) or a sequence homologous to said DNA.
or the recombinant vector according to (20) or (21) into a cell.
and expressing the polypeptide according to any one of (1) to (8).
A method for controlling cell differentiation, mutual recognition, and migration. (78) A method for controlling the differentiation, mutual recognition, and migration of cells from blood cells, nerve cells, stem cells, or cancer cells.
(79) The method according to (77), wherein the cell is selected from the DNA according to (10) to (18) or a sequence homologous to said DNA.
or the recombinant vector according to (20) or (21) is introduced into the promyeloma.
and introducing the polypeptide into a blastocyst to express the polypeptide according to any one of (1) to (8).
A method for promoting differentiation of promyelocytic cells into granulocytic cells by using the method. The present invention is described in detail below. (1) Obtaining DNA encoding the polypeptide of the present invention, and
and oligonucleotide production GlcNAcβ1,3-galactosidase disclosed in JP 6-181759
Galβ1-transferase (hereinafter abbreviated as β3Gal-T1; also known as WM1)
GlcNAcβ1,3-galactose transfer involved in the synthesis of 3GlcNAc structure
It is an enzyme. From the gene database, BLAST [J. Mol. Biol.2
51, 403-410 (1990)], FASTA (Methods in E
nzymology,183, 63-69), FrameSearch [Com
Using a program such as that manufactured by Pugen, genes homologous to this enzyme gene were identified.
The possibility of encoding a protein with homology to this enzyme at the amino acid level
By searching for a gene, it is possible to obtain DNA encoding the polypeptide of the present invention.
Alternatively, information on the base sequence of a portion of the DNA can be obtained.
Examples include GenBank, EMBL, and Geneseq (Derwent Public Library).
You can use public databases such as the National Institute of Information and Communications Technology, or you can use private databases.
A database of such genes is also available.
The salt of a gene that may encode a protein with amino acid homology to
An example of the base sequence is the base sequence of rat cDNA shown in SEQ ID NO: 3.
In addition, the base sequence of human cDNA that has homology to the base sequence of SEQ ID NO: 3 is
A human EST sequence with GenBank number AI039637 can be mentioned.
These sequences are partial base sequences of the DNA encoding the polypeptide of the present invention.
Single-stranded cDNA or cDNA library prepared from various organs or cells
The resulting DNA was used as a template and polymerized using primers specific to the sequence obtained above.
Polymerase Chain Reaction
PCR (Molecular Cloning,
A Laboratory Manual, Second Edition, C
old Spring Harbor Laboratory Press (1
989) (hereinafter referred to as Molecular Cloning, 2nd Edition) and PCR P
protocols Academic Press (1990)
The presence of DNA encoding the polypeptide of the present invention can be detected by
Similarly, a DNA fragment encoding the polypeptide of the present invention can be obtained.
If the obtained DNA fragment is not full-length, the full-length cDNA can be obtained as follows.
The DNA fragment obtained above can be used as a probe to confirm the presence of the DNA.
By screening a cDNA library derived from the selected organ or cell,
Full-length cDNA can be obtained by this method. In addition, single-stranded cDNA or cDNA library containing the DNA can be obtained.
By performing 5' RACE and 3' RACE using the library as a template,
Obtain the 5'-end and 3'-end fragments of the cDNA having the corresponding sequence.
By ligating both fragments, a full-length cDNA can be obtained. Single-stranded cDNA derived from various organs or cells can be prepared by conventional methods or commercially available.
It can be prepared according to a kit. An example is shown below: Guanidinium thiocyanate phenol-
Chloroform method [Anal. Biochem.,162, 156-159 (19
87)]. If necessary, the total RNA is purified by deoxyribonucleic acid.
The sample was treated with clease I (Life Technologies) to remove any possible contamination.
For each of the total RNA obtained, oligo (
dT) primer or random primer using SUPERSCRIPT^T
M Preamplification System for First
Strand cDNA System (Life Technologies
Single-stranded cDNA is synthesized using a DNA synthesis kit (manufactured by the company).
Single-stranded cDNA prepared by the above method from the colon cancer cell line Colo205 and human gastric mucosa
Examples of cDNA libraries include NA. cDNA libraries can be prepared by standard methods.
As for the method of producing the gene, see Molecular Cloning, 2nd Edition and Current P
rotocols in Molecular Biology, John W.
Iley & Sons (1987-1997) (hereafter referred to as Current Protocol)
(abbreviated as "Rules in Molecular Biology") Supplements 1-38, etc.
The method described in the literature or commercially available kits, such as SuperScript Plasmid
System for cDNA Synthesis and Plasmid Cloning
SuperScript Plasmid System for cDNA
A Synthesis and Plasmid Cloning; GIBC
OBRL] and ZAP-cDNA synthesis kit [ZAP-cDNA
Synthesis Kit (manufactured by STRATAGENE)
cDNA libraries derived from various organs or cells are commercially available.
It can also be obtained by purchasing products that are sold in Japan. As a cloning vector for creating a cDNA library,
Any phage vector or plasmid that can replicate autonomously in E. coli K12 strain.
Any vector can be used. Specifically, ZAP Express [ST
RATAGENE, Strategies,5, 58 (1992)], pB
bluescript SK(-), pBluescript II SK(+)
[Nucleic Acids Research,17, 9494 (1989
) ], λZAP II (STRATAGENE), λgt10, λgt11
[DNA Cloning, A Practical Approach,1,
49 (1985)], λTriplEx (manufactured by Clontech), λExCe
pT7T318U (Pharmacia)
), pcD2 [Mol. Cell. Biol. ，3, 280 (1983)], p.
UC18 [Gene,33, 103 (1985)], pAMo [J. Biol.
Chem.,268, 22782-22787 (1993), alias pAMoPR
C3Sc (JP 05-336963)], pCXN2 [Gene,108, 1
93 (1991)]. As a host microorganism, any microorganism belonging to the Escherichia coli group can be used.
It is possible. Specifically,Escherichia coliXL1-Blue
MRF' (Stratagene, Strategies,5, 81 (19
92)Escherichia coli C600 [Genetics,
39, 440 (1954)],Escherichia coliY1088
[Science,222, 778 (1983)],Escherichia
coli Y1090 [Science,222, 778 (1983)],Es
Cherichia coli NM522 [J. Mol. Biol. ，166 , 1 (1983)],Escherichia coliK802 [J. Mo
l. Biol.,16, 118 (1966)],Escherichia co
li JM105 [Gene,38, 275 (1985)],Escheric
hia coliSOLR^TMStrain (manufactured by STRATAGENE)
andEscherichia coliLE392 (Molecular Clones
As a cDNA library, for example, a cDNA library prepared as follows is used.
Examples include poly(A) from human gastric mucosa.⁺RNA to cDNA synthesis system (cDNA
cDNA synthesis was performed using a cDNA synthesis system (GIBCO BRL).
A is synthesized and on both endsEcoRI-NotI-SalI adapter (Super
rChoice System for cDNA Synthesis;G
IBCO BRL) was added to the cloning vector λZAP II (
λZAP II/EcoRI/CIAP Cloning Kit, STRAT
(manufactured by AGENTE)EcoInserted into the RI site and Gigapack III Go
Packaging Extract (manufactured by STRATAGENE) was used.
Itin vitroBy packaging, the cDNA library
Alternatively, a commercially available cDNA library can be purchased and used. Based on the base sequence of the candidate gene identified through a database search,
Specific primers were designed to amplify the single-stranded cDNA or
PCR is performed using the cDNA library as a template. When an amplified fragment is obtained,
The fragment is subcloned into an appropriate plasmid.
The DNA fragments are purified as they are or after treatment with restriction enzymes or DNA polymerases using conventional methods.
The vector can be inserted into a vector as follows.
Bluescript SK(-), pBluescript II SK(+
) (both manufactured by STRATAGENE), pDIRECT [Nucleic
Acids Research,18, 6069 (1990)], pCR-Am
pSK(+) [STRATAGENE, Strategies,5, 62
64 (1992)], pT7Blue [manufactured by Novagen], pCR II [
Invitrogen Biotechnology,9, 657 (199
1)], pCR-TRAP [Genehunter], pNoTAT7 (5
'→3') and the like. By determining the base sequence of the subcloned PCR-amplified fragment, the target
The DNA fragments obtained are confirmed.
analytical methods, such as the dideoxy method of Sanger et al. [Proc. Na
tl. Acad. Sci. USA,74, 5463 (1977)] or 37
Base sequence analysis using a 3A DNA sequencer (manufactured by Perkin Elmer)
This can be determined using a cDNA analysis device. The DNA fragment is used as a probe for the cDNA library prepared above.
Colony hybridization or plaque hybridization (mono
By performing recursive cloning (Second Edition), β3Gal-T1 and
Obtain cDNAs that may encode proteins with homology at the amino acid level
As a probe, the DNA fragment can be used as an isotope or a dinucleotide.
Digoxigenin-labeled versions can be used.
The base sequence of the DNA obtained by the above method can be determined by directly analyzing the DNA fragment.
Or, after cleaving with an appropriate restriction enzyme, etc.,
The vector is then inserted into a vector by a conventional method, and the DNA is sequenced by a conventional method, e.g., by sequencing.
The dideoxy method of Sanger et al. [Poc. Natl. Acad. Sci.
ci. USA,74, 5463 (1977)] or DNA Sequencer 3
73A (Perkin Elmer), DNA Sequencer 377 (Pe
Elmer), DNA sequencer model 4000L (
The sequence is determined by analyzing the sequence using a base sequence analyzer such as a nucleotide sequence analyzer manufactured by LI-COR.
The DNA of the present invention obtained by this method can be, for example, the DNA represented by SEQ ID NO: 1.
or a polypeptide having an amino acid sequence represented by SEQ ID NO: 1.
A polypeptide encoding the amino acid sequence from 39th to 378th of the sequence
Specifically, the base sequence represented by SEQ ID NO: 2 can be
DNA having the base sequence of SEQ ID NO: 2, the bases 135 to 1268 of which are
DNA having the base sequence, or 249 to 1 of the base sequence represented by SEQ ID NO: 2
Examples include DNA having the 268th base sequence. The base sequence represented by SEQ ID NO: 2 is a sequence similar to the amino acid sequence represented by SEQ ID NO: 1.
The base sequence is derived from cDNA encoding a polypeptide having the sequence shown in SEQ ID NO: 2.
The nucleotide sequence from 135th to 1268th of the nucleotide sequence shown in
It is a base sequence of only the coding region, and 249 of the base sequence represented by SEQ ID NO: 2
The base sequence from base 1 to base 1268 defines the region of the polypeptide having glycosyltransferase activity.
It is a base sequence that encodes the nucleotide sequence from bases 135 to 1268 of the nucleotide sequence represented by SEQ ID NO: 2.
Examples of plasmids containing this DNA include pAMo-G6 and pCXN2-G.
Examples include pBS-G6 and pBS-G6. Generally, there are multiple genetic codes for one amino acid, so SEQ ID NO: 2
Even DNA having a base sequence different from that of the polypeptide of the present invention can be used.
If so, it is included in the DNA of the present invention. Furthermore, based on the information on the base sequence of the DNA of the present invention obtained by the above method,
The entire or any part of the base sequence of the DNA of interest is subjected to 15 to 5'
DNA containing a 30-base sequence and a sequence complementary to the 15-30 base sequence at the 3' end
DNA containing the sequence is used as a sense primer and an antisense primer,
c prepared from mRNA of cells expressing mRNA complementary to these DNAs
By performing PCR using DNA as a template, the DNA of the present invention and its function can be obtained.
The DNA used as a primer can be applied to
Applied Biosystems 380A,
It can be synthesized using a DNA synthesizer such as the 392 or 3900. Furthermore, based on the amino acid sequence of the polypeptide encoded by the DNA of the present invention,
The DNA of the present invention can also be produced by chemically synthesizing the DNA encoding the polypeptide.
A can be prepared. Chemical synthesis of DNA is carried out using the thiophosphite method.
A Shimadzu DNA synthesizer was used, and the phosphoramidite method was used.
- Using DNA synthesizers such as 380A, 392, and 3900 manufactured by Biosystems
It is also possible to obtain DNA having a sequence complementary to the base sequence of the DNA obtained by the above method.
By selecting DNA that hybridizes under stringent conditions with
, in the amino acid sequence represented by SEQ ID NO: 1, one or more amino acids are deleted or substituted
or a target DNA encoding a polypeptide consisting of an added amino acid sequence.
A can be obtained from other species (mouse, rat, cow, monkey, etc.).
Specifically, the homologous DNA of the organism can be cloned.
The DNA encoding the polypeptide of the present invention can be synthesized by any method, except for the method using a DNA synthesizer.
In the case of the present invention, the nucleic acid sequence is obtained as a double-stranded DNA and encodes the polypeptide of the present invention.
It consists of a base strand of DNA and a DNA having a sequence complementary to the base sequence of the DNA.
Both DNAs were separated by heating at 100°C for 5 minutes and then quickly cooling on ice.
The sequence complementary to the base sequence of the DNA encoding the polypeptide of the present invention can be
The DNA having the sequence encodes the polypeptide of the present invention isolated as described above.
The sense strand DNA is used as a template, and the 5-30 base sequence at the 3' end is complementary to the
DNA consisting of a specific sequence is used as a primer, and DNA polymerase is carried out in the presence of dNTP.
The DNA that can hybridize under stringent conditions can also be synthesized by carrying out a DNA synthesis reaction.
The DNA was used as a probe for colony hybridization and plaque hybridization.
Hybridization method or Southern blot hybridization method
Specifically, it refers to DNA obtained by using colonies or
Using a filter on which plaque-derived DNA was immobilized, 0.7 to 1.0 mol
Hybridization was carried out at 65°C in the presence of 1/L of sodium chloride, and then 0
1 to 2 times concentrated SSC solution (the composition of 1 times concentrated SSC solution is 150 mmol
15 mmol/L sodium chloride, 15 mmol/L sodium citrate)
List the DNA that can be identified by washing the filter at 65°C.
Hybridization is described in Molecular Cloning, 2nd Edition, Current
Protocols in Molecular Biology, DNA Cloning
1: Core Techniques, A Practical Appro
ach, Second Edition, Oxford University
(1995) and other methods.
Specific examples of DNA that can be searched include BLAST [J. Mol. Biol.,21
5, 403 (1990)] and FASTA (Methods in Enzyme
Logy,183, 63-98 (1990)], and other analysis software,
When calculated using the parameters (initial setting), the DNA obtained above was slightly different from
DNA having at least 60% or more homology, preferably 70% or more, more preferably
or 80% or more, more preferably 90% or more, particularly preferably 95% or more,
Most preferably, the DNA has a homology of 97% or more. The DNA and DNA fragments of the present invention obtained by the above-mentioned method can be used to perform molecular
or by the conventional method described in "Cloning", 2nd edition, etc., or by the base sequence information of the DNA.
A sense oligonucleotide having a partial sequence of the DNA of the present invention is synthesized by a DNA synthesizer from the information.
a partial sequence of a base sequence complementary to the base sequence of the DNA of the present invention;
and preparing oligonucleotides, such as antisense oligonucleotides, having the
The oligonucleotide can be a base sequence of the DNA of the present invention or a salt thereof.
A base sequence complementary to the base sequence is preferably 5 to 120 consecutive bases, more preferably 1
Examples of such DNA include DNA having the same sequence of 5 to 60 bases as the sequence
D having the same sequence as 5 to 120 consecutive bases in the base sequence represented by sequence number 2
NA or a sequence complementary to the base sequence represented by SEQ ID NO: 2,
The forward primer can be a DNA having the same sequence as base 0.
When used as a reverse primer, the melting temperature (Tm) and
The oligonucleotides described above in which the number of bases does not change drastically are preferred.
Specifically, oligonucleotides having the base sequences shown in SEQ ID NOs: 25 and 28, etc.
Furthermore, derivatives of these oligonucleotides (hereinafter referred to as oligonucleotide derivatives) can be used.
) can also be used as the oligonucleotide of the present invention. As the oligonucleotide derivative, the phosphate diester in the oligonucleotide
an oligonucleotide derivative in which a tert-butyl bond has been converted to a phosphorothioate bond;
The phosphodiester bond in the oligonucleotide is N3'-P5' phosphoramide.
oligonucleotide derivatives converted to ribonucleotide bonds,
Oligonucleotides in which the phosphate and phosphodiester bonds have been converted to peptide nucleic acid bonds
Derivatives, uracil in oligonucleotides is replaced with C-5 propynyluracil
The oligonucleotide derivatives, wherein uracil in the oligonucleotide is C-5 thiol,
Azole uracil substituted oligonucleotide derivatives, oligonucleotides
An oligonucleotide derivative in which cytosine in the
In the oligonucleotide, the cytosine is a phenoxazine-modified cytosine (phenoxazine-modified cytosine).
oxazine-modified cytosine-substituted oligonucleotides
Nucleotide derivatives, oligonucleotides in which ribose is 2'-O-propyl ribonucleotide
oligonucleotide derivatives substituted with bases or oligonucleotides
Oligonucleotide derivatives in which the ribose is replaced with 2'-methoxyethoxyribose
Conductors, etc. [Cell engineering,16, 1463 (1997)]. The polypeptide of the present invention encoded by the DNA of the present invention obtained as described above
For example, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is
, the amino acid sequence from the 39th to the 378th positions of the amino acid sequence represented by SEQ ID NO: 1
A polypeptide comprising one or more amino acid sequences
The amino acid sequence of the present invention is a sequence in which the amino acid of the present invention is deleted, substituted or added, and
Ceramide β1,3-N-acetylglucosaminyltransferase activity and paraglobulin
Polypeptides having β1,3-N-acetylglucosaminyltransferase activity
or having 60% or more homology with the amino acid sequence of the polypeptide.
The amino acid sequence is lactosylceramide β1,3-N-acetylglucosamine.
Transaminase activity and paragloboside β1,3-N-acetylglucosamine transferase
Examples include polypeptides having transferase activity. Polypeptides having the above amino acid sequence in which one or more amino acids are deleted,
Proteins with substituted and/or added amino acid sequences are described in Molecule.
ular Cloning, A Laboratory Manual, Sec.
ond Edition, Cold Spring Harbor Labor
tory, (1989) (hereinafter referred to as "Molecular Cloning, 2nd Edition")
, Current Protocols in Molecular Biol
ogy, John Wiley & Sons, (1987-1997) (hereinafter
Current Protocols in Molecular Biology (abbreviated as "Current Protocols in Molecular Biology"), N
ucleic acids research,10, 6487 (1982),
Proc. Natl. Acad. Sci. ,USA,79, 6409 (1982
), Gene,34, 315 (1985), Nucleic Acids Re.
search,13, 4431 (1985), Proc. Natl. Acad.
Sci USA,82, 488 (1985) and other methods.
For example, a D gene encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1 can be prepared using the
This can be achieved by introducing site-specific mutations into the NA.
The number of amino acids to be added is one to several, and the number is not particularly limited.
However, deletions, substitutions, or other modifications can be made by well-known techniques such as the site-directed mutagenesis method described above.
The number is as many as can be added, for example, 1 to several tens, preferably 1 to 20,
More preferably, it is 1 to 10, and even more preferably, it is 1 to 5. Furthermore, the polypeptide of the present invention includes a polypeptide having an amino acid sequence represented by SEQ ID NO: 1 and
The amino acid sequence shown in SEQ ID NO: 1 has a homology of 60% or more.
The homology with the sequence was determined using BLAST [J. Mol. Biol.,215, 403 (19
90)] and FASTA (Methods in Enzymology,183 , 63-69) using the default (initial setting) parameters.
When calculated, it is at least 60% or more, preferably 70% or more, and more preferably
Preferably, the content is 80% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably 80% or more.
Preferably, the homology is 97% or more.
Homologous polypeptides in other organisms (mouse, rat, cow, monkey, etc.)
Specifically, the rat polypeptide represented by SEQ ID NO: 3 can be mentioned.
The polypeptide of the present invention exhibits β1,3-N-acetylglucosaminyltransferase activity.
The presence of the polypeptide of the present invention can be confirmed by the method described below in (3). (2) Production of the Polypeptide of the Present Invention The DNA of the present invention obtained by the above method is expressed in host cells, and the polypeptide of the present invention is produced.
To produce polypeptides, use Molecular Cloning, 2nd Edition, Current
Protocols in Molecular Biology Supplement 1-38
etc. can be used. That is, a construct in which the DNA of the present invention is inserted downstream of a promoter of an appropriate expression vector can be used.
The recombinant vector is constructed and introduced into a host cell, thereby producing the present invention.
A transformant expressing the polypeptide is obtained and the transformant is cultured.
The polypeptide of the present invention can be produced by the above method. Host cells of interest include prokaryotic cells, yeast, animal cells, insect cells, plant cells, etc.
Any gene can be used as long as it can express the gene.
Individual organisms or plants can be used. Expression vectors are those that can replicate autonomously in the host cells or that are chromosomally replicated.
and suitable for transcription of the DNA encoding the polypeptide of the present invention.
When prokaryotes such as bacteria are used as host cells, the polypeptide of the present invention can be encoded by the promoter.
The DNA expression vector is capable of autonomous replication in prokaryotes and is also capable of replicating the promoter.
a promoter, a ribosome binding sequence, a DNA encoding the polypeptide of the present invention,
Preferably, the promoter is composed of a transcription termination sequence.
The gene may be included. Expression vectors include, for example, pBTrp2, pBTac1, and pBTac2.
(both commercially available from Boehringer Mannheim), pSE28
0 (Invitrogen), pGEMEX-1 (Promega),
pQE-8 (QIAGEN), pKYP10 (JP 58-110600)
, pKYP200 [Agric. Biol. Chem. ，48, 669 (198
4)], pLSA1 [Agric. Biol. Chem. ，53, 277 (19
89)], pGEL1 [Proc. Natl. Acad. Sci. USA,82 , 4306 (1985)], pBlue II SK(-) (STRATAGE
NE), pTrs30 (FERM BP-5407), pTrs32 (FE
RM BP-5408), pGHA2 (FERM BP-400), pGKA2
(FERM B-6798), pTerm2 (JP-A-3-22979, US46
86191, US4939094, US5160735), pKK233-2 (
pGEX (Pharmacia), pET System
System (Novagen), pSupex, pUB110, pTP5, pC1
94, pTrxFus (Invitrogen), pMAL-c2 (New
Examples of promoters include those manufactured by England Biolabs. Any promoter that can be expressed in host cells such as E. coli can be used.
For example,trpPromoter (Ptrp),lacPromo
Tar (Plac), P_LPromoter, P_RPromoters, etc.,
Promoters derived from, for example, SPO1 promoter, SPO2 promoter,
penP promoter, etc. Also, two Ptrp promoters in tandem
Promoter (Ptrpx2),tacpromoter,lacT7 promoter
,letArtificially engineered promoters such as the I promoter are also used.
Ribosome binding sequences include Shine-Dalgarno sequences.
rno) sequence and the start codon to an appropriate distance (e.g., 6 to 18 bases).
It is preferable to use a plasmid containing a transcription termination sequence. A transcription termination sequence is not necessarily required for expression of the DNA of the present invention, but it is preferably
It is desirable to place a transcription termination sequence directly downstream of the structural gene. Host cells include Escherichia, Serratia, Bacillus, Brevibacter,
genus Corynebacterium, genus Microbacterium, genus Pseudomonas, etc.
Microorganisms belonging to, e.g.Escherichia coliXL1-Blu
e.Escherichia coliXL2-Blue,Escheric
hia coliDH1,Escherichia coliMC1000
,Escherichia coliKY3276,Escherichia coliW1485,Escherichia coliJM109,E
scherichia coliHB101,Escherichia co
liNo. 49,Escherichia coliW3110,Esch
erichia coliNY49,Escherichia coliB
L21 (DE3),Escheriehia coli BL21(DE3)p
LysS,Escherichia coli HMS174 (DE3),Es
Cherichia coli HMS174(DE3)pLysS,Serr
ati a ficaria,Serratia Fonticola,Serr
ati a likefaciens,Serratia marcescen
s,Bacillus subtilis,Bacillus amyloli
quefaciens,Brevibacterium ammoniagen
es,Brevibacterium immariophilumATCC
14068,Brevibacterium saccharolyticum ATCC14066,Corynebacterium glutamic acid m ATCC13032,Corynebacterium glutamic
um ATCC14067,Corynebacterium glutami
cum ATCC13869,Corynebacterium acetoa
cidophilum ATCC13870,Microbacterium
ammoniaphilum ATCC15354,Pseudomonas
sp. D-0110. The recombinant vector can be introduced by introducing DNA into the host cells as described above.
Any method can be used, for example, electroporation [Nuc
leic acids res. ，16, 6127 (1988)], calcium
The method using ions [Proc. Natl. Acad. Sci. USA,69,
2110 (1972)], protoplast method (JP-A-63-248394), G
ene,17, 107 (1982) and Mol. Gen. Genet. , 168,
111 (1979). When yeast strains are used as host cells, the expression vector can be, for example, Y
Ep13 (ATCC37115), YEp24 (ATCC37051), YCp
50 (ATCC37419), pHS19, pHS15, etc.
Any promoter can be used as long as it can be expressed in a yeast strain.
For example, the PHO5 promoter, the PGK promoter, the GAP promoter,
tar, ADH promoter, gal 1 promoter, gal 10 promoter
-, heat shock protein promoter, MFα1 promoter, CUP1 promoter
Examples of host cells include promoters of Saccharomyces, Schizosaccharomyces, Kluyveromyces, and the like.
Examples of yeast strains include those belonging to the genera Trichomyces, Trichosporon, and Siwaniomyces.
Specifically,Saccharomyces cerevisiae ,Schizosaccharomyces pombe,Kluyverom
yes lactis,Trichosporon pullulans,S
chwanniomyces alluviusThe recombinant vector can be introduced by any method that introduces DNA into yeast.
Any of these methods can be used, for example, electroporation [Methods
．． Enzymol. ，194, 182 (1990)], spheroplast method [P
roc. Natl. Acad. Sci. USA,84, 1929 (1978)]
, lithium acetate method [J. Bacteriol.,153, 163 (1983)]
, Proc. Natl. Acad. Sci. USA75, 1929 (1978
) and the like. When animal cells are used as host cells, the expression vector can be, for example, p
cDNAI/Amp (Invitrogen), pcDNAI (Funakoshi)
pCDM8 [Nature,329, 840 (1987), manufactured by Funakoshi Co., Ltd.
], pAGE107 [JP-A-3-22979; Cytotechnology,
3, 133 (1990)], pREP4 (Invitrogen), pAG
E103 [J. Biochemistry,101, 1307 (1987)],
pAMo [J. Biol. Chem. ，268, 22782 (1993)], p.
AMoA [J. Biol. Chem. ，268, 22782 (1993)], p.
Examples include AS3-3 (JP 2-227075). Any promoter that can be expressed in animal cells can be used.
For example, immediate endothelial cell death (IE) of cytomegalovirus (human CMV) can be
the promoter of the early gene, the early promoter of SV40,
Moloney Murine leukemia virus
Leukemia Virus long terminal repeat promoter
- (Long Terminal Repeat Promoter), Retro
Viral promoters, heat shock promoters, SRα promoters,
Alternatively, a metallothionein promoter may be used.
The enhancer of the CMV IE gene may be used together with the promoter. Any animal cell into which DNA can be introduced can be used as the host cell.
Cells can also be used. For example, mammalian cells include human, monkey, and mouse cells.
Cells from mouse, rat, guinea pig, or mink can be used.
These include mouse myeloma cells, rat myeloma cells, and mouse hybrid
tumour cells, Chinese hamster ovarian (CHO) cells (ATCC CR
L-9096, ATCC CCL-61), BHK cells, African green monkey kidney
Human liver cells, Namalwa cells (ATCC CRL-1432),
or Namalwa KJM-1 cells, human embryonic kidney cells, human leukemia cells, H
BT5637 (JP 63-299), human colon cancer cell line, etc.
Mouse myeloma cells include SP2/0 and NSO, and rat myeloma cells include
YB2/0 and other human embryonic kidney cells, HEK293 and 293
[J. Gen. Viol.36, 59-72 (1977)], etc., human leukemia cells
Examples of the cells include BALL-1, and African green monkey kidney cells include COS-1 (A
TCC CRL-1650), COS-7 (ATCC CRL-1651),
Examples of colon cancer cell lines include HCT-15. For the purpose of producing therapeutic protein drugs, mammalian cells, especially
It is preferable to use CHO cells as hosts. The recombinant vector can be introduced by any method, including introducing DNA into animal cells.
Any of these methods can be used, for example, electroporation [Cytot
technology,3, 133 (1990)], calcium phosphate method (Patent Publication No.
2-227075), DEAE dextran method (Yodosha Biomanual Series
Size 4,16), lipofection method [Proc. Natl. Acad. Sci.
.USA,84, 7413 (1987)], microinjection method (Yoshito
Biomanual Series 4,36), Adenovirus method (Yodosha Bio
Manual Series 4,43), vaccinia virus (Yodosha Biomanufacturer
Series 4,59) Retroviral Vectors (Yodosha Biomanual Series)
Size 4,74) etc. can be used. When insect cells are used as hosts, for example, baculovirus express
Prescription Vectors: A Laboratory Manual (Baculovir
us Expression Vectors, A Laboratory M
annual, W. H. Freeman and Company, New York
k (1992)], Molecular Biology: A Laboratory Manual
Molecular Biology, A Laboratory Man
ual), Current Protocols in Molecular Biology
Assembly 1-38 (Current Protocols in Molecules)
ular Biology), Bio/Technology,6, 47 (19
The polypeptide can be expressed by the method described in (88) etc. That is, a recombinant gene transfer vector and a baculovirus are co-transfected into insect cells.
After obtaining the recombinant virus in the insect cell culture supernatant, the recombinant virus was further
The vector can be used to infect insect cells and express the polypeptide. Examples of gene transfer vectors used in this method include pVL13
92 (Pharmingen), pVL1393 (Pharmingen)
pBlueBacIII (manufactured by Invitrogen) and the like.
An example of a baculovirus is a virus that infects insects in the family Mythodidae.
Autographa californica nuclear polyhedrosis virus (A
utographa californica nuclear polyhe
drossis virus), etc. can be used. Insect cells include:Spodoptera frugiperdaovarian cells
,Trichoplusia niovarian cells, cultured cells derived from silkworm ovaries, etc.
It can be used.Spodoptera frugiperdaovarian cells
Examples include Sf9 and Sf21 (baculovirus expression vectors).
Laboratory Manual, etc.Trichoplusia nieggs
The nest cells were High5 (also known as BTI-TN-5B1-4, Invitrogen
As cultured cells derived from silkworm ovaries,Bombyx MoriN
4. The above-mentioned recombinant gene transfer vector into insect cells to prepare a recombinant virus.
Examples of methods for co-transfection of the vector and the baculovirus include the calcium phosphate method.
(JP-A-2-227075), lipofection method [Proc. Natl. Ac
ad. Sci. USA,84, 7413 (1987)] and the like can be mentioned.
. In addition, using a method similar to that used to introduce DNA into animal cells,
NA can also be introduced, for example, by electroporation [Cytote
chnogy,3, 133 (1990)], calcium phosphate method (Patent Publication No. 2-2
27075), lipofection method [Proc. Natl. Acad. Sci.
USA,84, 7413 (1987)]. When plant cells or plant individuals are used as hosts, known methods (tissue culture
,20(1994), Tissue Culture,21(1995), Trends in Bi
otechnology,15, 45 (1997)].
The promoter used for gene expression is one that can be expressed in plant cells.
Any of these can be used, for example, cauliflower mosaic virus (Ca
Examples of promoters include the 35S promoter of the rice MV and the rice actin 1 promoter.
In addition, corn alcohol is inserted between the promoter and the gene to be expressed.
By inserting intron 1 of the acetylcholine dehydrogenase gene, the gene expression efficiency can be improved.
Host cells include potato, tobacco, corn, rice, rapeseed, and
Plant fibers such as ginseng, soybeans, tomatoes, wheat, barley, rye, alfalfa, and flax
Examples of methods for introducing a recombinant vector include transfection of plant cells with D
Any method for introducing NA can be used, for example, Agrobacterium
Lithium (Agrobacterium) (JP 59-140885, JP 6
0-70080, WO94/00977), electroporation method [Cyt
otechnology,3, 133 (1990), JP-A-60-251887
], a method using a particle gun (gene gun) (Patent No. 2606856, Patent
2517813). Plant cells and organs into which genes have been introduced can be mass-cultured using a jar fermenter.
In addition, by redifferentiating the transgenic plant cells,
It is also possible to create plant individuals into which genes have been introduced (transgenic plants).
The polypeptides of the present invention can also be produced using animal individuals.
Methods of Knowledge [American Journal of Clinical Nu
trition,63, 639S (1996), American Journal
al of Clinical Nutrition,63, 627S (199
6), Bio/Technology,9, 830 (1991) ],
The polypeptide of the present invention can be produced in an animal into which the gene has been introduced. Any promoter that can be expressed in animals can be used.
For example, the α-casein promoter, which is a mammary gland cell-specific promoter, can be used.
β-casein promoter, β-lactoglobulin promoter, whey acid promoter
A recombinant vector incorporating DNA encoding the polypeptide of the present invention is preferably used.
Transformants derived from microorganisms, animal cells, or plant cells containing the
The polypeptide is produced and accumulated by culturing the strain according to the method, and the polypeptide is extracted from the culture.
The polypeptide can be produced by collecting the transformant. When the transformant is an animal or plant, it can be raised and cultivated according to conventional methods.
and cultivating the animal or plant to produce and accumulate the polypeptide, and extracting the polypeptide from the animal or plant.
The polypeptide can be produced by collecting the polypeptide. In other words, in the case of an animal, for example, a non-human transgenic animal carrying the DNA of the present invention can be produced.
The recombinant DNA is then used to generate the polypeptide of the present invention.
The polypeptide is produced and accumulated in the animal, and then extracted from the animal.
The polypeptide of the present invention can be produced by the above method.
Examples of such animal products include milk (Japanese Patent Application Laid-Open No. 63-309192) and eggs.
In the case of individual plants, for example, transgenic plants carrying the DNA of the present invention can be used.
and growing the polypeptide of the present invention encoded by the recombinant DNA in the plant.
The polypeptide of the present invention can be obtained by synthesizing and accumulating the polypeptide in the plant and collecting the polypeptide from the plant.
The polypeptide-producing transformant of the present invention can be produced from prokaryotes such as E. coli or yeasts.
In the case of nucleocytic organisms, the medium in which these organisms are cultured contains a carbon source that can be assimilated by the organism,
Any medium containing nitrogen sources, inorganic salts, etc. that can efficiently cultivate transformants is acceptable.
Either a natural or synthetic medium may be used. The carbon source may be any that can be utilized by the transformant, such as glucose,
Fructose, sucrose, molasses containing these, starch or starch
Carbohydrates such as hydrolysates, organic acids such as acetic acid and propionic acid, ethanol, propane,
Alcohols such as ethanol are used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, and ammonium acetate.
ammonium salts of various inorganic and organic acids, such as ammonium and ammonium phosphate,
Other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep
Liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria and
and its digested products can be used. Inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate,
ammonium sulfate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate
, calcium carbonate, etc. can be used. Cultivation is carried out under aerobic conditions such as shaking culture or submerged aeration and agitation culture. Cultivation temperature
The temperature is preferably 15 to 40°C, and the incubation time is usually 16 to 96 hours.
The pH is adjusted by adding an inorganic or organic acid or alkali.
The culture is carried out using solutions such as urea, calcium carbonate, and ammonia. If necessary, antibiotics such as ampicillin and tetracycline may be added during the culture.
It may also be added to the medium. Transformed with an expression vector that uses an inducible promoter.
When culturing such microorganisms, an inducer may be added to the medium as needed.
Good. For example,lacMicroorganisms transformed with promoter-based expression vectors
When culturing
) etc.,trpCultivating microorganisms transformed with promoter-based expression vectors
When the transformant for producing the polypeptide of the present invention is an animal cell, the cell may be cultured.
The medium used was the commonly used RPMI 1640 medium [The Journal of
al of the American Medical Association
on,199, 519 (1967)], Eagle's MEM medium [Science
e.122, 501 (1952)], DMEM medium [Virology,8, 3
96 (1959)], 199 medium [Proceedings of the Society of
Society for the Biological Medicine,73 , 1 (1950)] or a medium containing fetal bovine serum or the like added to these media.
The culture is usually carried out at pH 6-8, 30-40°C, and 5% CO₂Under conditions such as in the presence of 1 to
The culture period is 7 days. If necessary, antibiotics such as kanamycin or penicillin may be added to the medium during the culture period.
A medium for culturing transformants obtained using insect cells as host cells is generally
TNM-FH medium (Pharmingen), Sf-900
II SFM medium (GIBCO BRL), ExCell 400, ExCell
ell405 (both manufactured by JRH Biosciences), Grace'
s Insect Medium [Nature,195, 788 (1962)
The culture is preferably carried out at a pH of 6 to 7 and a culture temperature of 25 to 30°C.
The culture time is usually 1 to 5 days. If necessary, gentamicin may be added during the culture.
Antibiotics such as thiamin may be added to the medium. Transformants obtained using plant cells as hosts can be stored as cells or as plant cells.
The transformant can be cultured by differentiating it into a cell or organ.
The commonly used Murashige and Skoog (MS) medium, White
(White) medium, or these media containing auxin, cytokinin, etc.,
A hormone-added medium can be used. The culture is usually carried out at a pH of 5 to 9, 2
The culture is carried out at 0 to 40°C for 3 to 60 days.
Antibiotics such as cysteine and hygromycin may be added to the medium. In addition to expressing the full-length polypeptide, the β1,3
- a partial polypeptide containing a region having N-acetylglucosaminyltransferase activity
Glycosyltransferases are generally expressed as type II membrane proteins.
It has a cytoplasmic topology, and the N-terminal cytoplasmic region consists of several to several tens of amino acids.
A membrane-binding region with a highly soluble amino acid sequence, a stem region consisting of several to several dozen amino acids,
the stem region, and most of the remaining C-terminal portion, including the catalytic domain.
The remaining C-terminal part, including the stem and catalytic domains, is transported to the Golgi apparatus.
The boundary between the stem and catalytic domains is thought to be exposed to the lumen.
We will create a polypeptide with the same structure and examine how much deletion is necessary to lose activity.
On the other hand, the knowledge about the stem region and catalytic region is
By comparing the amino acid sequence with that of similar glycosyltransferases, the stem region and catalytic
It is also possible to predict the region. The polypeptide of the present invention having the amino acid sequence set forth in SEQ ID NO: 1 has an N-terminal
A cytoplasmic domain consisting of 14 amino acids, followed by a hydrophobic domain consisting of 18 amino acids.
a membrane-binding domain containing at least 12 amino acids, a stem domain containing at least 12 amino acids, and a catalytic domain.
It can be predicted that the remaining majority of the C-terminal portion is composed of the 45th position.
The polypeptide containing the amino acid sequence from position 378 is thought to contain the catalytic domain.
The stem region is similar to other β1,3-N-acetylglucosaminyltransferases and β1,3-glycosyltransferases.
Comparison of amino acid sequence homology with lactosyltransferase and other β1,3
-N-acetylglucosaminyltransferase and β1,3-galactosyltransferase
Specifically, the results are estimated based on findings regarding the area of the sintered body.
For example, the sequence number can be estimated based on the findings disclosed in US Pat.
a secretory polypeptide comprising amino acids 36 to 378 of SEQ ID NO: 1, or
The secretory polypeptide containing amino acids 39 to 378 of sequence number 1 is β
Has 1,3-N-acetylglucosaminyltransferase activity. The full-length polypeptide or β1,3-N-acetylglucosaminyltransferase
A partial polypeptide containing an active region (catalytic region) can be expressed by any method other than direct expression.
In accordance with the method described in Molecular Cloning, 2nd Edition,
It can also be expressed as a protein or a fusion protein.
Proteins include β-galactosidase, protein A, and protein A Ig.
G-binding domain, chloramphenicol acetyltransferase, poly(Ar
g), poly(Glu), protein G, maltose binding protein, glutathione
S-transferase, polyhistidine chain (His-tag), S peptide
, DNA-binding protein domain, Tac antigen, thioredoxin, green fluorescent protein
fluorescent protein (GFP), FLAG peptide, and any antibody
Examples include epitopes of [Akio Yamakawa, Experimental Medicine,13, 469-474 (
1995)]. Methods for producing the polypeptide of the present invention include a method for producing it in host cells,
There are two methods: secretion outside the host cell, or production on the outer membrane of the host cell.
The method can be appropriately selected depending on the host cell to be used and the structure of the polypeptide to be produced.
When the polypeptide of the present invention is produced inside a host cell or on the outer membrane of a host cell,
, the method of Paulson et al. [J. Biol. Chem.,264, 17619 (19
89)], the method of Lowe et al. [Proc. Natl. Acad. Sci. USA,8
6, 8227 (1989), Genes Develop. ，4, 1288 (1
990)], or as described in JP-A-05-336963, WO94123021, etc.
By applying the above method, the polypeptide can be actively secreted outside the host cell.
That is, a gene containing the active site of the polypeptide of the present invention can be produced using genetic engineering techniques.
By expressing the polypeptide with a signal peptide added before it,
The polypeptide of the present invention can be actively secreted outside the host cell. Specifically, a polypeptide having an amino acid sequence thought to include a catalytic site
By adding a signal peptide before the polypeptide of the present invention and expressing it,
It is believed that the peptide can be actively secreted outside the host cell.
Between the signal peptide and the polypeptide containing the catalytic domain, or between the polypeptide containing the catalytic domain
A tag for purification and detection can also be added to the C-terminus of the polypeptide. Tags for purification and detection include β-galactosidase, protein A, and proteinase.
IgG binding region of clonal antibody A, chloramphenicol acetyltransferase
, poly(Arg), poly(Glu), protein G, maltose binding protein
, glutathione S-transferase, polyhistidine chain (His-tag)
, S peptide, DNA binding protein domain, Tac antigen, thioredoxin,
Green fluorescent protein (GFP), FLAG peptide, and
and epitopes of any antibody [Akio Yamakawa, Experimental Medicine,13, 46
9-474 (1995)]. Also, dihydrofolic acid was synthesized according to the method described in JP-A-2-227075.
It is also possible to increase production by using a gene amplification system that uses a reductase gene, etc.
Another method for producing the polypeptide of the present invention is to use the DNA of the present invention.in v
itroThere is a method for producing it using a transcription/translation system.in vitroTranscription and Translation Systems
This refers to the transcription of DNA into mRNA and the transcription of mRNA into genomic DNA using a cell-free system.
It is a system that produces polypeptides by translating DNA into proteins.
A cell-free system capable of producing a desired polypeptide from a target DNA or a target mRNA.
Any stem can be used.
For example, rabbit reticulocyte lysate
locyte lysate) and wheat germ lysate (Wheat Germ Lysate)
Examples include systems using methyl methyl acrylate.in vitroTranscription and translation systems are
A kit is commercially available from the company, and polypeptides can be extracted relatively easily using the commercially available kit.
Commercially available kits include, for example, In Vitro
Express^TM Translation Kit (STRATAGEN
The polypeptide of the present invention can be produced by a known method [J. Biology
molecular NMR,6, 129-134, Science,242 , 1162-1164, J. Biochem. ，110, 166-168 (19
91)in vitroIt can also be produced using a transcription-translation system.
The polypeptide of the present invention is extracted from the culture of the transformant for producing the polypeptide of the present invention.
For isolation and purification, a conventional enzyme isolation and purification method can be used. For example,
The polypeptide of the present invention is dissolved in the cells of the transformant for producing the polypeptide of the present invention.
If the cells accumulate in a solution state, the cells in the culture can be centrifuged.
The cells were collected, washed, and then subjected to ultrasonic homogenization using a French press, a Menton-Gauze
The cells are disrupted using a homogenizer, dynomill, etc. to obtain a cell-free extract. The cell-free extract is centrifuged to obtain a supernatant, which is then subjected to solvent extraction, sulfur extraction, etc.
The salting-out method by Yasu et al., desalting method, precipitation method using organic solvents, diethylaminoethyl (DE
AE) Resins such as Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation)
anion exchange chromatography using S-Sepharose FF (
Cation exchange chromatography using resins such as those manufactured by Pharmacia
Hydrophobic cloning using resins such as butyl sepharose and phenyl sepharose
chromatography, gel filtration using molecular sieves, affinity chromatography
methods such as the chromatographic method, chromatofocusing method, and electrophoretic methods such as isoelectric focusing.
A purified preparation can be obtained by using the same method. Furthermore, if the polypeptide is expressed by forming an insoluble body within the cell,
After collecting the cells, they were crushed and centrifuged to obtain a precipitate.
After recovering the polypeptide by the method, the insoluble body of the polypeptide is degraded.
The solubilized solution is prepared by adding a polypeptide denaturant or a polypeptide denaturant-free solution.
The peptide denaturant is diluted to a concentration that is low enough to prevent denaturation of the polypeptide.
After the polypeptide is reconstituted into its normal three-dimensional structure, it is subjected to the same single-step purification as above.
A purified preparation can be obtained by isolation and purification methods. If the polypeptide is secreted outside the cells, the culture can be purified by techniques such as centrifugation.
The soluble fraction is then extracted from the cell-free extract.
A purified preparation of the polypeptide is obtained by the same method as that for isolation and purification from the supernatant.
This is also possible using conventional methods for purifying glycosyltransferases (Methods in Enzymol
ogy,83, 458). The polypeptide of the present invention can also be produced as a fusion protein with another protein.
Then, affinity chromatography was performed using a substance that has affinity for the fused protein.
It can also be purified using roughage [Akio Yamakawa, Experimental Medicine,13, 469
-474 (1995)]. For example, the method of Rowe et al. [Proc. Natl. Aca.
d. Sci. USA,86, 8227 (1989), Genes Develo
p.,4, 1288 (1990)], JP-A-05-336963, JP-A-06-
The polypeptide of the present invention was reacted with Protein A according to the method described in JP-A-823021.
It is produced as a fusion protein and subjected to affinity cloning using immunoglobulin G.
The polypeptide of the present invention can be purified by chromatography.
The protein was produced as a fusion protein with the FLAG peptide, and subjected to immunohistochemistry using anti-FLAG antibodies.
The product can be purified by affinity chromatography [Proc. Natl.
l. Acad. Sci. USA,86, 8227 (1989), Genes D.
development.,4, 1288 (1990)]. Furthermore, affinity chromatography using antibodies against the polypeptide itself was performed.
The polypeptides of the present invention can also be purified using the Fmoc method (fluorenylmethyloxycarbonyl).
by chemical synthesis methods such as the t-Boc method (t-butyloxycarbonyl method)
It can also be manufactured by Advanced Chemtech.
ed ChemTech, Perkin-Elmer, Pharmacia Bio
Protein Technology Instrument (Protein Technology Instrument) manufactured by Tech Co., Ltd.
Technology Instruments, Synthesizer Vega (Sy
PerSeptive, manufactured by Nthecell-Vega
Chemical synthesis can also be performed using peptide synthesizers manufactured by Shimadzu Corporation or other companies. The structure of the purified polypeptide of the present invention can be analyzed using methods commonly used in protein chemistry.
For example, protein structure analysis for gene cloning (Hirano Hisashi, Tokyo University of Science)
This can be carried out by the method described in the publication "Gakudojin Publishing, 1993." (3) Measurement of the activity and use of the polypeptide of the present invention Cells prepared from a transformant harboring an expression vector for the polypeptide of the present invention.
The extract, or the polypeptide of the present invention isolated and purified from the transformant or its culture.
Using a peptide as an enzyme sample, the enzyme was assayed by a known method [J. Biol. Chem.
,268, 27118 (1993), J. Biol. Chem. ，267, 23
507 (1992), J. Biol. Chem. ，267, 2994 (1992
), J. Biol. Chem. ，263, 12461 (1988), Jpn. J
．． Med. Sci. Biol. ，42, 77 (1989), FEBS Lett.
．462, 289 (1999), J. Biol. Chem. ，269, 147
30-14737 (1994), J. Biol. Chem. ，267, 2994
(1992), Anal. Biochem. ，189, 151-162 (199
0), J. Biol. Chem. ，273, 433-440 (1998)]
Based on this, the β1,3-N-acetylglucosaminyltransferase activity of the polypeptide of the present invention
Furthermore, by measuring with various receptor substrates,
The acceptor substrate specificity of the polypeptide of the present invention can be examined. When a cell extract is used as the enzyme sample, the glycosyltransferase contained in the host cell itself can be examined.
In order to eliminate the influence of the enzyme activity, the DNA encoding the polypeptide of the present invention is not included.
The cell extract of the transformant into which the control vector was introduced was used as a control.
The β1,3-N-acetylglucosaminyltransferase activity of the control
β1,3-N-acetylglucosaminyltransferase activity was increased compared to that of the control.
Therefore, the polypeptide encoded by the DNA of the present invention contained in the transformant is β1
It can be said that they have Galβ1,3-N-acetylglucosaminyltransferase activity. Cells expressing multiple Galβ1,3-N-acetylglucosaminyltransferases
In the case of cells and tissues, enzymatic analysis using cell or tissue extracts as the enzyme source revealed that the expressed
By identifying each of the Galβ1,3-N-acetylglucosaminyltransferases
and each of their Galβ1,3-N-acetylglucosaminyltransferases
The enzymatic properties of each strain cannot be clarified.
By purifying the polypeptide of the present invention by
To clarify the enzymatic properties of alβ1,3-N-acetylglucosaminyltransferase
Examples of assay methods are shown below. (i) Methods using fluorescently labeled oligosaccharides as acceptor substrates Oligosaccharides fluorescently labeled by 2-aminobenzoation or pyridylamination
The reaction was carried out using UDP-GlcNAc as a sugar donor and the reaction solution was
Analyze by high performance liquid chromatography (HPLC).
The strand substrate was prepared using SIGMA 2AB glycan labeling kit.
It was used in accordance with the kit instructions.
Fluorescent labeling by pyridyl amination can be carried out according to the standard method [Agr
ic. Biol. Chem. ，54, 2169 (1990)].
The sugar donor UDP-GlcNAc was added compared to the absence of it.
The peak that increases when the product is added is the product peak. The amount of the product is measured from its fluorescence intensity.
The ratio of the product to the added acceptor substrate was determined as β1,3-N-acetylglucosamine.
The product was identified by its HPLC retention time.
, standard (the oligosaccharide of the acceptor substrate used in the reaction is linked to the reducing end by a β1,3 bond)
The retention time of the labeled oligosaccharide (with N-acetylglucosamine attached) coincides with that of the labeled oligosaccharide (with N-acetylglucosamine attached).
This can be done using the fact that the nucleotide sequence is the same as that of the nucleotide sequence. (ii) Method using unlabeled oligosaccharides as the acceptor substrate The reaction is carried out in the same manner as in (i) using unlabeled oligosaccharides as the acceptor substrate, and the nucleotide sequence is then quantified.
The reaction mixture was analyzed by high performance anion exchange chromatography instead of LC.
The donor UDP-GlcNAc concentration increases when added compared to when not added.
The peak was used as the product peak and the amount of product was measured.
The ratio of the product to the β1,3-N-acetylglucosaminyltransferase activity was determined.
The ratio of the product to the added acceptor substrate was calculated as β1,3-N-acetylglucosamine
The product was identified by high-performance anion exchange chromatography.
The elution time of the standard (reducing end of the oligosaccharide acceptor substrate used in the reaction)
oligosaccharides with N-acetylglucosamine attached to the ends via β1,3 bonds)
This can be done by using the fact that the reaction time coincides with the reaction time as an indicator. (iii) Method using glycolipids as acceptor substrates Using glycolipids as acceptor substrates, UDP-[¹⁴C]GlcNAc as a glycosyl donor
Glycolipids are extracted from the reaction mixture by reverse phase chromatography and then separated by silica gel.
The plate is developed using a thin layer chromatography (TLC) plate.
The product is detected and quantified by measuring the radioactivity.
The ratio of the resulting product to the total product is taken as the β1,3-N-acetylglucosaminyltransferase activity.
The product was identified by using the Rf value of the product as a standard (the reduced value of glycolipid used as the acceptor substrate).
A glycolipid with a structure in which N-acetylglucosamine is attached to the original end via a β1,3 bond)
This can be done using the Rf value as an indicator of whether it matches the Rf value. Furthermore, the polypeptide of the present invention can be used to determine whether it has a sugar chain synthesis in vivo.
Based on the description in (2), it is possible to analyze whether or not the protein is involved in the formation of animal cells.
The transformed cells are prepared by introducing an expression vector containing the DNA of the present invention into the cells and culturing the cells.
The transformed cells are then transformed to express the polypeptide of the present invention.
(Poly-N-acetyllactosamine sugar chain, sialyl Lewis a sugar chain, sialyl Lewis
After fluorescent staining using antibodies or lectins that specifically bind to the sugar chains,
Fluorescence Activated Cell Sorter
Activated Cell Sorter (hereinafter abbreviated as FACS)
The amount of glycans bound by antibodies or lectins is analyzed using a control vector that does not contain DNA encoding the polypeptide of the present invention.
Compared with the vector-introduced transformed cells, poly-N-acetyllactosamine sugars
By confirming the increase in the amount of chain, it is possible to confirm that the polypeptide encoded by the DNA of the present invention is
β1,3-N-acetyllactosamine, which is involved in the synthesis of poly-N-acetyllactosamine sugar chains
It has been found that it has poly-N-acetyllactosamine transferase activity. Antibodies and lectins that recognize poly-N-acetyllactosamine glycans include poly
Any substance that recognizes N-acetyllactosamine sugar chains can be used.
For example, an antibody that recognizes poly-N-acetyllactosamine sugar chains can be used.
As a body, it specifically binds linear poly-N-acetyllactosamine sugar chains (i antigens).
and a branched poly-N-acetyllactosamine sugar.
Anti-I antibody that specifically recognizes the I chain (I antigen) [J. Biol. Chem.,254, 3221 (1979)], a protein that recognizes poly-N-acetyllactosamine sugar chains
Cutin includes pokeweed mitogen (abbreviated as PWM),Lyc
OPERSICON esculentumagglutinin (LEA and
(abbreviated)Datura stramonium agglutinin (DSA
(abbreviated as "J. Biol. Chem.,282, 8179
-8189 (1987), J. Biol. Chem. ，259, 6253-62
60 (1984), J. Biol. Chem. ，262, 1602-1607 (
1987), Carbohydr. Res. ，120, 187-195 (198
3), Carbohydr. Res. ，120, 283-292 (1983),
Glycoconjugate J.7, 323-334 (1990)]. The polypeptides of the present invention are useful for in vivo synthesis of sugar chains of glycoconjugates such as glycoproteins and glycolipids.
In the above method, the fact that the gene is involved in in vivo synthesis is due to the fact that the gene is involved in in vivo synthesis of the gene of the transformed cell.
During the culture, a glycosyltransferase inhibitor specific to each glycoconjugate was added to inhibit the synthesis of glycosyltransferase.
Cells in which synthesis is inhibited can be identified by FACS analysis.
The transformed cells are cultured in the presence of an inhibitor specific to the synthesis of O-linked glycans of the target protein.
After that, the transformed cells were incubated with an antibody that recognizes poly-N-acetyllactosamine sugar chains.
When fluorescent staining was performed using the α-acetyllactosamine derivative and analyzed by FACS, poly-N-acetyllactosamine sugars were detected.
The amount of O-linked sugar chains is reduced, so that the polypeptide of the present invention can be easily synthesized by the method described above.
It is known that the glycan is involved in the synthesis of poly-N-acetyllactosamine sugar chains in the glycan.
. Glycosylation inhibitors specific to the glycans of glycoconjugates include the O-linked glycans of glycoproteins.
Benzyl-α-GalNAc, an inhibitor of the synthesis of N-linked glycans of glycoproteins
Swainsonine, a mannosidase II inhibitor that acts as an inhibitor of the synthesis of glycolipid glycosidases
D-PD, an inhibitor of glucosylceramide synthase, acts as an inhibitor of the synthesis of hydroxylase chains.
MP(D-threo-1-phenyl-2-decanoylamino-
3-morpholino-1-propanol), etc. Furthermore, transformed cells obtained by introducing an expression vector for the polypeptide of the present invention into animal cells
and a control vector not containing DNA encoding the polypeptide of the present invention.
Glycolipids were extracted from the transformed cells and the composition of both glycolipids was analyzed by TLC plate.
By analyzing and comparing using the same, it was found that the polypeptide of the present invention binds to the sugar chain of glycolipids.
The extraction of glycolipids and the analysis of their composition were carried out by known methods.
[Shujunsha, Cell Engineering Special Issue, Glycobiology Experimental Protocol, Anal.
Biochem.,223, 232 (1994)].
Glycolipids developed on TLC plates were stained with orcinol or with specific glycan structures.
It was detected and quantified by immunostaining using an antibody that binds to
The Rf value of the glycolipid is compared with that of the corresponding glycolipid.
Paragloboside (Galβ1-4GlcNA) was isolated using an antibody that recognizes the cis-aminosamine structure.
cβ1-3Galβ1-4Glc-ceramide) and neolactohexaosylceramide
Lamid (Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1
When immunostained with the polypeptide of the present invention,
Transfected cells expressing the peptide showed a significant increase in the number of paradigms compared with control transfected cells.
Increased amounts of loboside and neolactohexaosylceramide
The polypeptide of the present invention is a compound comprising paragloboside and neolactohexaosylceramide.
It has been found that the polypeptide of the present invention is involved in the synthesis of inflammatory diseases, cancer, cancer metastasis, etc.
The present invention can be used as a pharmaceutical for treating, preventing, and/or diagnosing diseases associated with altered expression of
The pharmaceutical containing the polypeptide of the present invention can be administered alone as a drug.
Although it is possible to administer other drugs, it is usually recommended to administer one or more pharmacologically acceptable drugs.
and mixed with the above carriers and formulated by any method well known in the art of pharmaceutical formulation.
The route of administration is determined based on the optimal route for treatment.
It is desirable to use the most effective one, which can be administered orally, into the mouth, into the respiratory tract, or directly.
Examples of parenteral administration include enteral, subcutaneous, intramuscular and intravenous administration.
Forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, and injections.
Examples include injections, ointments, tapes, etc. Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, and powders.
Liquid preparations such as emulsions and syrups include:
Water, sucrose, sorbitol, fructose and other sugars, polyethylene glycol, propylene glycol
glycols such as ethylene glycol, oils such as sesame oil, olive oil, and soybean oil, p-
Preservatives such as hydroxybenzoates, strawberry flavor, peppermint
It can be produced using flavors such as cinnamon and other flavorings as additives.
Powders and granules contain excipients such as lactose, glucose, sucrose, and mannitol, and starch.
disintegrants such as cellulose, sodium alginate, magnesium stearate, talc, etc.
Lubricants, polyvinyl alcohol, hydroxypropyl cellulose, gelatin, etc.
Additives include binders, surfactants such as fatty acid esters, and plasticizers such as glycerin.
Preparations suitable for parenteral administration include injections, suppositories, sprays, etc.
For example, injections may contain a carrier such as a salt solution, a glucose solution, or a mixture of both.
Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats, or carboxylic acids.
The spray is prepared by spraying the protein itself or the oral cavity and
It does not irritate the respiratory tract mucosa and disperses the protein into fine particles, making it easy to absorb.
Specific examples of carriers include lactose and glycerin.
Depending on the properties of the protein and the carrier used, it may be used in the form of an aerosol, a dry powder, or the like.
In addition, these parenteral preparations can be used as oral preparations with additives.
The ingredients exemplified above can also be added. The dosage or frequency of administration should be determined based on the desired therapeutic effect, administration method, treatment period, age,
Although it varies depending on body weight, the usual daily dose for adults is 1 μg/kg to 100 mg/kg.
Furthermore, the lactosylceramide β1,3-N-acetylglucosamine conversion of the present invention
A polypeptide having transferase activity and a DNA encoding said polypeptide are obtained.
This will enable the following functional analyses and applications: (i) Molecular biological techniques, or knockout mice and transgenics
(ii) Functional analysis of the polypeptide of the present invention using mice, etc. (ii) Development of the polypeptide of the present invention and DNA encoding said polypeptide
(iii) Analysis of the distribution of the polypeptide of the present invention and the DNA encoding the polypeptide
(iv) Analysis of the relationship between expression and various diseases. (iv) Expression of the polypeptide of the present invention and DNA encoding said polypeptide.
(v) Expression of the polypeptide of the present invention and DNA encoding the polypeptide.
Identification of cell types and differentiation stages using this as an indicator. (vi) Development of the polypeptide of the present invention and DNA encoding said polypeptide.
Searching for compounds that enhance or inhibit the enzymatic activity of the polypeptide of the present invention
(vii) Synthesis of useful sugar chains using the polypeptide of the present invention. The following describes the use of the polypeptide of the present invention and the DNA encoding said polypeptide.
Specific examples are given below. (4) N-acetylglucosamine is attached to a galactose residue via a β1,3 bond.
Production and Use of Sugar Chains Having a Structure and Glycoconjugates Containing the Sugar Chains The polypeptide of the present invention binds to galactose at the non-reducing end of the sugar chain of an acceptor substrate.
The activity of transferring N-acetylglucosamine to the β1,3 bond of the β1,
Therefore, the present invention
The peptide can be used as a sugar chain synthesis agent.
Galactose, N-acetyllactosamine (Galβ1-4GlcNAc), G
alβ1-3GlcNAc or lactose (Galβ1-4Glc), ii)
Galactose, N-acetyllactosamine, Galβ1-3GlcNAc or
an oligosaccharide having a lactose structure at the non-reducing end, and iii) galactose;
N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure
The polypeptide of the present invention is a complex carbohydrate having the above i at the non-reducing end.
galactose present at the non-reducing end of the sugar chain of the acceptor substrate selected from
It has the enzyme activity to transfer N-acetylglucosamine to the residue via a β1,3 bond.
Glycoconjugates include glycoproteins, glycolipids, proteoglycans, glycopeptides,
Glycosides in which sugar chains are bound to lipopolysaccharides, peptidoglycans, and steroid compounds
Galactose, N-acetyllactosamine,
A compound having a Galβ1-3GlcNAc or lactose structure at the non-reducing end
Examples of glycoconjugates include lactosylceramide and paragloboside.
N-acetylglucosamine is attached to a galactose residue via a β1,3 bond.
Examples include GlcNAcβ1-3Gal structure, GlcNAcβ1-3Galβ1
-4GlcNAc structure, GlcNAcβ1-3Galβ1-3GlcNAc structure
, GlcNAcβ1-3Galβ1-4Glc structure, (Galβ1-4GlcN
Acβ1-3)_nGalβ1-4GlcNAc structure (n is an integer of 1 or more),
is (Galβ1-4GlcNAcβ1-3)_nGalβ1-4Glc structure (n is
(an integer of 1 or more), etc. Details are provided below. (i) Using a transformant into which DNA encoding the polypeptide of the present invention has been introduced.
Method for producing sugar chains Traits derived from microorganisms, animal cells, plant cells, and insect cells obtained in (3) above
A transformant selected from the transformants is cultured in a culture medium, and N-acetyl
Sugar chains with a structure in which glucosamine is attached to galactose residues via a β1,3 bond
or complex carbohydrates containing said sugar chains, specifically GlcNAcβ1-3Galβ1
-4Glc-ceramide, lacto-series glycolipid (Galβ1-3GlcNAcβ1-3
Glycolipids having Galβ1-4Glc-ceramide as the backbone), neolacto-series sugars
Lipid (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide
glycolipid having a GlcNAcβ1-3Gal structure as the backbone, a sugar having a GlcNAcβ1-3Gal structure, Glc
Sugars having a cNAcβ1-3Galβ1-4GlcNAc structure, GlcNAcβ
Sugars having a 1-3Galβ1-3GlcNAc structure, GlcNAcβ1-3Ga
Sugars with a 1β1-4Glc structure (Galβ1-4GlcNAcβ1-3)_n
Sugars having a Galβ1-4GlcNAc structure and n being 1 or greater, and (Galβ
1-4GlcNAcβ1-3)_nGalβ1-4Glc structure and n is 1 or more
A sugar chain consisting of a sugar selected from a group consisting of certain sugars, or a complex sugar containing said sugar chain
The sugar chain or glycoconjugate is then collected from the culture.
The sugar chain or glycoconjugate can be produced by culturing the transformant in accordance with (3) above.
The polypeptide and any recombinant glycoprotein (e.g., a recombinant glycoprotein for pharmaceutical use)
By simultaneously producing the recombinant saccharides (sugar chains) in a transformant capable of synthesizing the saccharide chains,
N-acetylglucosamine is linked to galactose residues in glycoproteins via a β1,3 bond
A sugar chain having an additional structure can be added. (ii) An animal or plant into which DNA encoding the polypeptide of the present invention has been introduced.
Method for producing glycans using individual animals Using the individual animals or plants obtained in (3) above, a method for producing glycans using the individual animals or plants obtained in (3) above is carried out in accordance with the method in (3) above.
N-acetylglucosamine is attached to the galactose residue via a β1,3 bond.
and producing a sugar chain having a structure attached to a structure or a complex carbohydrate having said sugar chain attached.
In other words, in the case of an animal, for example, a non-human transgenic animal carrying the DNA of the present invention can be
The animals were raised to have N-acetylglucosamine in a β1,3 bond.
sugar chain having a structure in which the sugar chain is attached to a galactose residue, or a complex sugar having the sugar chain attached thereto
Specifically, GlcNAcβ1-3Galβ1-4Glc-ceramide, lactate
Glycolipids (Galβ1-3GlcNAcβ1-3Galβ1-4Glc-sera
glycolipids having β1-4Glc as a backbone), neolacto-series glycolipids (Galβ1-4Glc
a glycolipid having NAcβ1-3Galβ1-4Glc-ceramide as the backbone),
Sugars having a GlcNAcβ1-3Gal structure, GlcNAcβ1-3Galβ1
-4GlcNAc structure, GlcNAcβ1-3Galβ1-3Glc
Sugars having a NAc structure, GlcNAcβ1-3Galβ1-4Glc structure
Sugars containing Galβ1-4GlcNAcβ1-3_nGalβ1-4GlcNAc
sugars having the structure (Galβ1-4GlcNAcβ1-3
)_nGalβ1-4Glc structure and n is 1 or more.
and causing the animal to produce and accumulate a sugar chain consisting of the sugars or a complex carbohydrate containing the sugar chain.
By collecting the product from the inside, it was found that N-acetylglucosamine was bound to the β1,3 bond.
sugar chains having a structure in which a galactose residue is attached to the sugar chain or complexes having the sugar chain attached
Carbohydrates can be produced. Examples of places where carbohydrates are produced and stored in the animal include the animal's milk and eggs.
In the case of individual plants, for example, transgenic plants carrying the DNA of the present invention can be used.
and N-acetylglucosamine was linked to galactose via a β1,3 bond in the plant.
The present invention produces and stores sugar chains having structures attached to sugar residues or complex carbohydrates to which the sugar chains are attached.
The product is then collected from the plant to produce N-acetylglucosamine.
A sugar chain having a structure in which galactose is attached to a galactose residue via a β1,3 bond, or said sugar chain
(iii) Method for Producing Glycoconjugates Using the Polypeptide of the Present Invention The polypeptide of the present invention obtained by the method described in (3) above can be used as an enzyme source.
In an aqueous medium, galactose monosaccharide, galactose residue at the non-reducing end
The acceptor group is a complex carbohydrate having a oligosaccharide or galactose residue at the non-reducing end of the sugar chain.
As a substance, a galactose residue or galactose present at the non-reducing end of the sugar chain
The reaction product in which N-acetylglucosamine is attached to a monosaccharide via a β1,3 bond is referred to as
It can be produced by the following methods: i) lactosylceramide (Galβ1-4Glc-ceramide) or paclitaxel
Lagloboside (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-
ramide), ii) galactose, N-acetyllactosamine (Galβ1-4G
lcNAc), Galβ1-3GlcNAc, Galβ1-3GalNAc, or
or lactose (Galβ1-4Glc), iii) galactose, N-acetyl
Lactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc
or an oligosaccharide having a lactose (Galβ1-4Glc) structure at the non-reducing end
, iv) galactose, N-acetyllactosamine, Galβ1-3GlcNA
c or a complex carbohydrate having a lactose structure at the non-reducing end
The present invention, which is obtained by the method described in (3) above, using at least one species as an acceptor substrate.
The polypeptide is used as an enzyme source, and the acceptor substrate, the enzyme source, and uridine-5'
-N-acetylglucosamine diphosphate (hereinafter referred to as UDP-GlcNAc)
is present in an aqueous medium, and the acceptor substrate galactose or
A reaction in which N-acetylglucosamine is attached to a galactose residue via a β1,3 bond
The reaction product is produced and accumulated, and the reaction product is collected from the aqueous medium.
The reaction product can be produced. The enzyme source is lacto-N-neotetraose (lacto-N-neotetr
aose, Galβ1-4GlcNAcβ1-3Galβ1-4Glc) as substrate
1 μmol of GlcNAcβ1-3Galβ1-4Gl per minute at 37°C.
The activity capable of producing cNAcβ1-3Galβ1-4Glc is 1 unit (
U) is 0.1 mU/l to 10,000 U/l, preferably 1 mU/l
It is used at a concentration of 1 to 1,000 U/L. Aqueous media include water, phosphates, carbonates, acetates, borates, citrates,
Buffer solutions such as Tris, alcohols such as methanol and ethanol, ethyl acetate
esters such as acetone, ketones such as acetamide, and amides such as acetamide.
In addition, the culture medium of the microorganism used as the enzyme source may be an aqueous medium.
The transformant obtained by the culture described in (2) above can be used as a
a culture medium of the non-human transgenic animal, a milk obtained from the non-human transgenic animal described in (2) above,
The aqueous medium may contain a surfactant, if necessary.
Alternatively, an organic solvent may be added. Surfactants include polyoxyethylene octadecylamine (e.g., nylon).
nonionic surfactants such as MEAN S-215 (manufactured by NOF Corporation); cetyl trimethicone;
Alkyl ammonium bromide and alkyl dimethyl benzyl ammonium chloride
Cationic surfactants such as iodide (e.g., Cation F2-40E, manufactured by NOF Corporation)
agents, anionic surfactants such as lauroyl sarcosinate, alkyldimethyl
tertiary amines such as N-aminolamine (e.g., Tertiary Amine FB, manufactured by Nippon Oil & Fats Co., Ltd.);
-acetylglucosamine has a structure in which it is attached to a galactose residue via a β1,3 bond
Any sugar chain or sugar chain-attached glycoconjugate that promotes the production of the sugar chain is also usable.
The surfactant may be any of the surfactants, and one or more of them may be used in combination.
It is usually used at a concentration of 0.1 to 50 g/l. Organic solvents include xylene, toluene, aliphatic alcohols, acetone, and acetic acid.
Ethyl, etc., and is usually used at a concentration of 0.1 to 50 ml/l.
As for -GlcNAc, in addition to commercially available products, reaction products produced using the activity of microorganisms, etc.
The UDP-Glc may be purified from the reaction mixture.
NAc can be used at a concentration of 0.1 to 500 mmol/L. In the above, galactose, N-acetyllactosamine (Galβ1-4G
lcNAc), Galβ1-3GlcNAc, or lactose (Galβ1-
In addition to the above, examples of oligosaccharides having a Galβ4Glc structure at the non-reducing end include Galβ4Glc and Galβ5Glc.
1-3GalNAc, Galβ1-4GlcNAcβ1-3Galβ1-4Gl
c, Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc, Gal
β1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc, Ga
lβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4GlcNA
c, Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)G
lc, Galβ1-4GlcNAcβ1-3Galβ1-4 (Fucα1-3)
GlcNAc, Galβ1-3GlcNAcβ1-3Galβ1-4Glc, G
alβ1-3GlcNAcβ1-3Galβ1-4GlcNAc, Galβ1-
3GlcNAcβ1-3Galβ1-4 (Fucα1-3) Glc, Galβ1
-3GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc,G
alβ1-4GlcNAcβ1-3 (GlcNAcβ1-6) Galβ1-4G
lc, Galβ1-4GlcNAcβ1-3 (GlcNAcβ1-6) Galβ
1-4GlcNAc, Galβ1-4GlcNAcβ1-3 (Galβ1-4G
lcNAcβ1-6) Galβ1-4Glc, Galβ1-4GlcNAcβ1
-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc,G
alβ1-3GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4G
lc, Galβ1-3GlcNAcβ1-3 (GlcNAcβ1-6) Galβ
1-4GlcNAc, Galβ1-3GlcNAcβ1-3 (Galβ1-4G
lcNAcβ1-6) Galβ1-4Glc, Galβ1-3GlcNAcβ1
-3 (Galβ1-4GlcNAcβ1-6) Galβ1-4GlcNAc, or
or an oligosaccharide having one of these oligosaccharide structures at the non-reducing end of the sugar chain.
Examples include oligosaccharides, which have a galactose residue at the non-reducing end of the sugar chain.
The complex carbohydrates that can be used include those that have one of the above oligosaccharide structures as a non-reducing sugar chain.
Glycoconjugates containing terminal glycans or asialo complex type N-linked glycans
Specifically, for example, lactosyl
Ceramide (Galβ1-4Glc-ceramide) and paragloboside (Galβ1-
Examples of glycolipids include glycolipids such as β1-4GlcNAcβ1-3Galβ1-4Glc-ceramide.
The acceptor substrate can be used at a concentration of 0.01 to 500 mmol/L. MnCl₂inorganic salts such as β-mercapto
The reaction can be carried out in an aqueous solution.
In a medium, at a pH of 5 to 10, preferably 6 to 8, and at a temperature of 20 to 50° C., the
The sugar chains or glycoconjugates produced by the above method are then subjected to a known enzymatic or
A part of the sugar chain can be excised by chemical methods [Japanese Biochemical Society, ed., Digestive Chemistry
Experimental Lectures, Vol. 4, Research Methods for Complex Carbohydrates I and II, Tokyo Kagaku Dojin, (1986)
, Naoyuki Taniguchi, Akimi Suzuki, Kiyoshi Furukawa, Kazuyuki Sugawara, Supervisor, Glycobiology Experimental Program
Tocol, Shujunsha, (1996)]. The glycans to which N-acetylglucosamine is added obtained by the above method can also be used.
or a complex carbohydrate is used as an acceptor substrate, and (a) the acceptor substrate, (b) GlcNAcβ
1,4-galactosyltransferase, and (c) uridine-5'-diphosphate galactosyltransferase.
UDP-Gal is present in an aqueous medium,
β1,4 bond to the N-acetylglucosamine residue at the non-reducing end of the acceptor substrate
A reaction product having galactose added thereto is produced and accumulated, and the galactose is extracted from the aqueous medium.
The galactose is obtained by collecting the sugar chain or complex carbohydrate to which lactose is added.
It is possible to produce such sugar chains or glycoconjugates to which poly-N-acetyllactosamine sugar chains [(Galβ1-4GlcNAc
β1-3) structure is repeated two or more times] is GlcNAcβ1,4-galactosidase
Alternating between β1,3-N-acetylglucosaminyltransferase and Galβ1,3-N-acetylglucosaminyltransferase activity
It is known that GlcNAcβ1 is synthesized by acting on
4-galactosyltransferase and Galβ1,3-N-acetylglucosamine of the present invention
Using a polypeptide having phosphotransferase activity,in vitroPoly-N-
Acetylactosamine sugar chains can be synthesized. That is, using the polypeptide of the present invention as an enzyme source, (a) GlcNAcβ1,
4-galactosyltransferase, (b) i) lactosylceramide (Galβ1-4G
lc-ceramide) or paragloboside (Galβ1-4GlcNAcβ1-3
Galβ1-4Glc-ceramide), ii) galactose, N-acetyllacto
Galβ1-4GlcNAc, Galβ1-3GlcNAc or
galactose (Galβ1-4Glc), iii) galactose, N-acetyllactose
tosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc, or
Oligosaccharides having a lactose (Galβ1-4Glc) structure at the non-reducing end, iv.
) galactose, N-acetyllactosamine, Galβ1-3GlcNAc or
is a complex carbohydrate having a lactose structure at the non-reducing end, and v)
(c) an acceptor substrate selected from the resulting sugar chains or glycoconjugates;
N-acetyllactosamine phosphate (UDP-GlcNAc), and (d) cucumber
UDP-5'-diphosphate galactose (UDP-Gal) in an aqueous medium.
Therefore, in the aqueous medium, poly-N-acetyllactosamine is attached to the non-reducing end of the acceptor substrate.
A reaction product having a poly-N-glycosylation chain attached thereto is produced and accumulated, and the poly-N-glycosylation product is extracted from the aqueous medium.
By collecting glycans or glycoconjugates to which acetyllactosamine sugar chains have been added,
and the sugar chain or complex carbohydrate to which the poly-N-acetyllactosamine sugar chain is attached.
Furthermore, it is possible to produce the GlcNAcβ1,4-galactosyltransferase gene and the Ga
A polypeptide having 1β1,3-N-acetylglucosaminyltransferase activity is
By co-expressing the DNA encoding the poly-N-acetyllactosine
GlcN sugar chains or complex carbohydrates to which such sugar chains are attached can be produced.
Acβ1,4-galactosyltransferase is expressed in most cells, so G
A polypeptide having alβ1,3-N-acetylglucosaminyltransferase activity
Poly-N-acetyltransferase can also be produced by expressing only the encoding DNA in cells.
Lactosamine sugar chains or glycoconjugates with such sugar chains can be produced. (iv) Uses of Various Sugar Chains or Glycoconjugates The various sugar chains or glycoconjugates produced in the above manner can be used, for example.
The following applications are possible: Human milk contains lacto-N-neotetraose (Galβ1-4GlcNAc
β1-3Galβ1-4Glc) and lacto-N-tetraose (Galβ1-3
GlcNAcβ1-3Galβ1-4Glc), or a structure having these as the backbone
It is known that there are various oligosaccharides with different structures [Acta Paedia
iatrica,82, 903 (1993)]. These oligosaccharides commonly contain GlcN.
The oligosaccharides have a poly-N-acetylglucosamine structure.
Oligosaccharides containing cetyllactosamine sugar chains are also included. These oligosaccharides include:
Helps protect infants from viral and microbial infections and neutralizes toxins
It is also believed to have the activity of promoting the growth of bifidobacteria, which are beneficial intestinal bacteria.
On the other hand, the types of oligosaccharides present in the milk of animals such as cows and mice are also known.
The majority of these oligosaccharides in human milk are lactose.
Almost none exist. The above oligosaccharides contained in human milk, or milk containing them, are efficiently
If the lactobacillus of the present invention can be efficiently produced, it will be very useful in industry.
Polypeptides with β1,3-N-acetylglucosaminyltransferase activity
The peptides are involved in the synthesis of the above oligosaccharides in human milk in the mammary gland.
oligosaccharides that may be effective in fighting infections and promoting the growth of beneficial bifidobacteria
It may be possible to use it for the production of Poly-N-acetyllactosamine sugar chains contribute to protein stabilization.
Therefore, it is possible to artificially add poly-N-acetyllactosamine sugar chains to any protein.
By adding it, proteins can be stabilized.
The rate of kidney clearance of proteins increases with increasing effective molecular weight.
Therefore, it is possible to artificially add poly-N-acetyllactosamine to any protein.
By adding a sugar chain and increasing the effective molecular weight, the rate of clearance from the kidney is improved.
Furthermore, the poly-N-acetate can be used to reduce the blood glucose concentration and increase the blood stability.
By adding tyrrolactosamine sugar chains, any protein can be targeted to specific cells.
Targeting is also possible. (5) Use of the DNA or oligonucleotide of the present invention Regarding the use of the DNA, oligonucleotide, or derivatives thereof of the present invention,
The details are described below. (i) Quantification of the expression level of DNA encoding the polypeptide of the present invention Also, the DNA of the present invention or the above-mentioned oligonucleotides prepared from said DNA.
The expression level of the gene encoding the polypeptide of the present invention is quantified or the gene is expressed using the
It is possible to quantify the expression level of mRNA encoding the polypeptide of the present invention or to detect structural changes in the polypeptide of the present invention.
Methods for detecting structural changes in DNA or mRNA encoding a polypeptide include:
For example, (a) Northern blotting (b)in situationHybridization
(c) quantitative PCR/real-time PCR
) method, (d) differential hybridization method, (e) DNA
(f) RNase protection assay, etc.
Examples of specimens to be analyzed using the above method include cultured cells, various tissues, and serum.
, biological samples such as saliva, DNA obtained from the transformant described in (3), mRNA
A or total RNA is used. Hereinafter, the mRNA and total RNA are referred to as sample-derived
The tissue obtained from the biological sample is called RNA.
Alternatively, RNA isolated as a phage section can be used. In the Northern blot method, RNA from a sample is separated by gel electrophoresis and then immersed in nylon 1000.
The DNA is transferred onto a support such as a filter, and a labeled probe prepared from the DNA of the present invention is used.
By carrying out hybridization and washing, the polypeptide of the present invention can be obtained.
By detecting a band that specifically binds to the mRNA encoding the m
Changes in the expression level and structure of RNA can be detected.
When carrying out the hybridization, the mRNA in the sample-derived RNA and the probe are hybridized in a stable manner.
Incubate under conditions that form a lid. To prevent false positives,
Hybridization and washing steps can be performed under highly stringent conditions.
These conditions include temperature, ionic strength, base composition, probe length, and
The concentration of formamide is determined by a number of factors, such as:
The method is described in Molecular Cloning, 2nd Edition. The labeled probes used in the Northern blotting method can be prepared by known methods (e.g., nick
translation, random priming, or kinesing)
A chiral isotope, biotin, a fluorescent group, a chemiluminescent group, etc., is added to the base sequence of the DNA of the present invention.
DNA having a complementary base sequence, a partial fragment of said DNA of 100 bases or more, or
can be prepared by incorporating the DNA sequence into an oligonucleotide designed from the DNA sequence.
The amount of the bound labeled probe reflects the expression level of the mRNA.
The amount of the mRNA expression can be quantified by quantifying the amount of the labeled probe.
In addition, by analyzing the labeled probe binding site, structural changes in the mRNA can be detected.
The above-mentioned labeled probe and tissue obtained from a living body can be stored in paraffin or cryostat.
Hybridization and washing using isolated stat sections
Carry out the processin situationBy hybridization method, the mRNA
The expression level can be detected.in situationIn the hybridization method,
To prevent false positives, hybridization and washing steps should be performed at high stringency.
It is desirable to carry out the analysis under gentle conditions. These conditions include temperature, ionic strength, and base composition.
is determined by a number of factors, including the composition, length of the probe, and formamide concentration.
These factors are described, for example, in Molecular Cloning, 2nd Edition.
Quantitative PCR, differential hybridization, or D
The detection of the mRNA by the NA microarray method/DNA chip method etc. is carried out by
RNA or cDNA synthesized from the RNA using reverse transcriptase
Hereinafter, this cDNA will be referred to as sample-derived cDNA.
In quantitative PCR, a random primer or an oligo dT primer can be used. In quantitative PCR, the cDNA derived from the sample is used as a template, and the DNA of the present invention is
A primer designed based on the base sequence of the DNA of the present invention (the base sequence of the DNA of the present invention)
DNA having the same sequence as the continuous 5 to 120 bases of the sequence and the DNA of the present invention
The base sequence has the same sequence as the base sequence of 5 to 120 consecutive bases that are complementary to the base sequence of the
PCR is performed using a set of oligonucleotides of DNA containing the
A DNA fragment derived from mRNA encoding the polypeptide of the present invention is amplified.
The amount of amplified DNA fragments reflects the expression level of the mRNA.
Glyceraldehyde-3-phosphate dehydrogenase
-3-phosphate dehydrogenase, hereinafter abbreviated as G3PDH
By placing DNA encoding the mRNA as an internal control,
It is possible to quantify the amount of A. Furthermore, the amplified DNA fragments can be subjected to gel electrophoresis.
By further separating the mRNA, it is possible to know the change in the structure of the mRNA.
It is possible to use appropriate primers that specifically and efficiently amplify the target sequence.
Suitable primers are those that do not cause inter-primer or intra-primer bonds.
It specifically binds to the target cDNA at the annealing temperature and then binds to the target under denaturing conditions.
The amplified DNA fragment can be designed based on conditions such as deviation from the cDNA.
Quantification of the amplified product must be performed during the PCR reaction when the amplified product is increasing exponentially.
In such a PCR reaction, the amplified DNA fragments produced in each reaction are
This can be determined by collecting the DNA and quantitatively analyzing it using gel electrophoresis. Real-time PCR method [Junko Stevens, Experimental Medicine (Special Edition),1
5, 46-51 (1997)] is based on the same principle as the above quantitative PCR, and
Man probe and forward primers labeled with two types of fluorescent dyes
PCR was performed using the primer and reverse primer, and the amount of amplified DNA fragment was
The amount of emitted fluorescence can be detected in real time. The DNA of the present invention is immobilized on a filter using cDNA derived from a sample as a probe.
- or hybridization to a substrate such as a glass slide or silicon
By carrying out the above steps, the expression of mRNA encoding the polypeptide of the present invention can be confirmed.
The method based on this principle is called Defa
Differential hybridization method [Trends in Genetic
s,7, 314-317 (1991)] and DNA microarray method/DNA chip
The genomic DNA fragmentation method [Genome Research,6, 639-645 (1996)] and
Both methods involve culturing actin or Glycine macrophages on a filter or substrate.
Immobilization of an internal control such as 3PDH allows for a clearer separation between the control and target samples.
In addition, the difference in the expression of the mRNA between the control sample and the target sample can be accurately detected.
Based on the RNA from the target specimen, labeled cDNA was produced using different labeled dNTPs.
Two labeled cDNA probes are synthesized and placed on a single filter or substrate.
By simultaneously hybridizing the two antibodies, the expression level of the mRNA can be accurately quantified.
In the RNase protection assay, a T7 promoter is first attached to the 3' end of the DNA of the present invention.
After binding a promoter sequence such as a promoter or SP6 promoter, a labeled rN
Using RNA polymerase in the presence of TPin vitroTranscription is performed with
The labeled antisense RNA is synthesized by the above steps.
The NA binds to the sample-derived RNA to form an RNA-RNA hybrid.
After that, it was digested with RNase, and the RNA fragments protected from digestion were separated by gel electrophoresis.
The resulting bands are then quantified to determine the identity of the polynucleotides of the present invention.
The expression level of mRNA encoding the peptide can be quantified. The above-mentioned detection methods can be used to detect the presence of the protein of the present invention in diseases such as inflammatory diseases, cancer, and cancer metastasis.
Use in the detection or diagnosis of diseases associated with altered expression of genes encoding proteins
When carrying out the above detection or diagnosis, it is necessary to
and patients with diseases associated with altered expression of the DNA encoding the polypeptide of the present invention.
DNA, mRNA, or total RNA obtained from patients with HIV and healthy individuals was examined.
The DNA, mRNA or total RNA is used as a sample of a patient or a healthy individual.
Various tissues, serum, saliva, and other biological samples, or cells obtained from the biological samples, are used for testing.
It can be obtained from primary cultured cells cultivated in vitro in an appropriate medium.
The gene encoding the polypeptide of the present invention was analyzed using samples from patients and healthy individuals.
The expression levels of the gene in patients and healthy individuals are measured and compared using the detection methods described above.
The expression level range of the gene in the subject's sample is determined.
By comparing the expression level of the gene, it is possible to detect or treat diseases associated with altered expression of the gene.
In addition to the polypeptide of the present invention, two types of Galβ1,3-N-acetyltransferases have already been identified.
Although the glucosaminyltransferase has been cloned, the specific Galβ1,3-
To detect the expression of N-acetylglucosaminyltransferase, the base sequence of the gene
Sequence-based detection methods (e.g., Northern hybridization and PCR)
The DNA of the present invention can be used to synthesize two enzymes that have already been cloned.
It is now possible to distinguish between these two and accurately examine their expression. A specific example of quantifying expression levels is given below. The expression of lactosylceramide β1,3-N-acetylglucosaminyltransferase
It regulates the differentiation of blood cells and the mutual recognition and migration of nerve cells.
It is thought that abnormal expression of this enzyme or a decrease or increase in activity due to mutation of this enzyme may be the cause.
It is suspected that various diseases may occur due to the effects of the polypeptide of the present invention.
and diagnosing the above-mentioned various diseases by quantifying the expression level of DNA encoding the above.
For example, the treatment for myeloid leukemia and lymphocytic leukemia is different, so
It is believed that a method for accurately distinguishing between these two types of leukemia would be extremely useful in medical practice.
In myeloid cell lines, lactosylceramide β1,3-N-acetylglucosamine
Transaminase activity is detected in lymphoid cell lines, whereas activity is not detected in lymphoid cell lines.
Therefore, the gene encoding the polypeptide of the present invention is not present in myeloid leukemia cells.
The gene encoding the polypeptide of the present invention is expressed in lymphocytic leukemia cells.
It is assumed that the gene is not expressed.
, total RNA or cDNA is prepared, and a base sequence complementary to the base sequence of the DNA of the present invention is obtained.
DNA having the sequence or a partial fragment of said DNA of 100 bp or more, or
The above oligonucleotides were designed based on the DNA sequence of
A gene encoding the polypeptide of the present invention is prepared by hybridization or PCR.
By quantifying the expression levels of genes involved in the disease, it is possible to distinguish between myeloid leukemia and lymphocytic leukemia.
(ii) Obtaining and identifying the promoter region and transcriptional regulatory region of the DNA of the present invention Furthermore, the DNA of the present invention can be used as a probe to detect transcriptional regulation by known methods [
New Cell Engineering Experimental Protocols, edited by the Cancer Research Department, Shujunsha (1993)
, the promoter region and transcriptional regulatory region of the DNA can be obtained and identified.
Using chromosomal DNA isolated from mouse, rat, or human cells and tissues,
The DNA or oligonucleotide of the present invention is then added to the prepared genomic DNA library.
Plaque hybridization was performed using the cDNA fragment (especially the 5' end of the cDNA) as a probe.
By screening using methods such as DNA amplification, the DNA of the present invention can be identified.
Promoter regions and transcriptional regulation in mouse, rat, or human genomic DNA
The base sequence of the obtained genomic DNA and the cDNA can be obtained.
The exon/intron structure of the DNA is revealed by comparing the base sequences.
It is possible to use a similar method to test this method in other non-human mammals.
The promoter region and transcriptional regulatory region of the DNA can be obtained. Currently, the sequences of many human chromosomal genes with unknown functions are registered in databases.
Therefore, the sequence of the human cDNA encoding the polypeptide of the present invention and the data
By comparing the sequences of the human chromosomal genes registered in the database,
Identification of the human chromosomal gene encoding the polypeptide and elucidation of its structure
If a chromosomal gene sequence that matches the cDNA sequence is registered,
By comparing the sequence of the NA with the sequence of the chromosomal gene, the polypeptide of the present invention
Promoter region, exon and intron structure of the chromosomal gene encoding
The promoter region can be used to encode the polypeptide of the present invention in mammalian cells.
All promoter regions and transcriptional regulatory regions involved in the transcription of the gene being monitored
The transcriptional regulatory region includes a gene encoding the polypeptide of the present invention.
Enhancer sequences that enhance basal transcription and silencer sequences that attenuate it.
For example, white blood cells, nerve cells, tracheal cells, and lung cells.
, colon cells, placental cells, neuroblastoma cells, glioblastoma cells, colon cancer
cells, lung cancer cells, pancreatic cancer cells, gastric cancer cells, and leukemia cells.
Examples of the promoter region and transcriptional control region that function in the
The promoter and transcriptional regulatory region can be used in the screening method described below.
This method is useful for analyzing the transcriptional regulatory mechanism of the gene. (iii) Detection of mutations and polymorphisms in the DNA encoding the polypeptide of the present invention The novel β1,3-N-acetylglucosaminyltransferase of the present invention can be used to detect poly-N-acetylglucosaminyltransferases.
It is also involved in the synthesis of sialic acid sugar chains, and is therefore thought to be involved in the synthesis of sialic acid sugar chains in leukocytes.
Lewis x sugar chains and cancer-related sugar chains in cancer cells (sialyl Lewis x sugar chains, sialyl Lewis x sugar chains,
Regarding the synthesis of Lewis a sugar chains, sialyl Lewis c sugar chains, and dimeric Lewis a sugar chains
Therefore, by examining the mutations and polymorphisms of the DNA of the present invention,
It is believed that the present invention can be used to diagnose inflammatory diseases, cancer, or cancer metastasis, or to predict the prognosis of cancer.
The DNA or polypeptide of the present invention can also be used to detect polymorphisms and mutations in the DNA of the present invention.
By investigating the relationship between the mutations and diseases in the organs where the DNA is expressed,
Therefore, it is possible to diagnose other diseases such as functional abnormalities of the gene based on polymorphisms and mutations of the gene.
It can also be used for diagnostics. A method for detecting mutations in the DNA of the present invention is described below. In order to evaluate the presence or absence of disease-causing mutations in the DNA of the present invention,
The most definitive test for this is to directly compare DNA from a control population with DNA from diseased patients.
Specifically, the present invention aims to compare the results of mutations in the DNA encoding the polypeptides of the present invention.
Human biological samples such as tissues, serum, saliva, or other samples from patients with certain diseases and healthy individuals.
a primary culture cell established from the biological sample is collected, and the biological sample or the primary culture cell is
DNA is extracted from the cells (hereinafter, this DNA is referred to as sample-derived DNA).
Using the sample-derived DNA as a template,
The polypeptide of the present invention can be isolated by PCR using primers designed based on the above.
Alternatively, the DNA encoding the peptide is amplified from the biological sample or the primary culture.
By performing similar PCR using cDNA derived from cultured cells as a template,
In this way, the DNA encoding the desired polypeptide can be amplified.
The amplified DNA obtained from the patient was compared with the amplified DNA obtained from a healthy individual.
As a method of comparison, the presence or absence of mutations can be determined by
Direct base sequence analysis: DNA with wild-type sequence and DNA with mutation
Methods for detecting heteroduplexes formed by hybridization with (see below)
etc. can be used. In addition, mutations that cause the above-mentioned diseases in the DNA encoding the polypeptide of the present invention
As a method for detecting whether a DNA strand has a wild-type allele or a mutant allele,
Heteroduplexes formed by hybridization with DNA strands carrying opposite alleles
Methods for detecting heteroduplexes can be used. Methods for detecting heteroduplexes include (a) polyacrylamide gel electrophoresis.
Heteroduplex detection method [Trends Genet.,7, 5 (1991)]
(b) Single-strand conformation polymorphism analysis [Genomics,16, 32
5-332 (1993)], (c) chemical cleavage of mismatches (CCM, chem
cleavage of mismatches) [Human
Molecular Genetics (1996), Tom Stracha
n and Andrew P. Read (BIOS Scientific
Publishers Limited)], (d) enzymatic cleavage of mismatches
[Nature Genetics,9, 103-104 (1996)], (e
) Denaturing gel electrophoresis [Mutat. Res.,288, 103-112 (19
93)]. (a) Heteroduplex detection method using polyacrylamide gel electrophoresis Using sample-derived DNA or sample-derived cDNA as a template,
By performing PCR using primers designed based on the base sequence described in
The primers are:
The amplified DNA (derived from the patient) is designed to amplify DNA of 1 bp or less.
DNA, or a mixture of amplified DNA from patients and amplified DNA from healthy individuals
) is converted into single-stranded DNA by heat denaturation, and then the temperature is gradually lowered to
The double-stranded DNA is then subjected to polyacrylamide gel electrophoresis.
If a heteroduplex is formed, it is more likely to be formed than a homoduplex without the mutation.
The migration rate of the ATP is also slower and they can be detected as extra bands.
The separation is better when using a solvent such as Hydro-link or MDE.
If searching for fragments smaller than p, insertions, deletions, and most single-base substitutions can be detected.
Heteroduplex analysis is a step similar to single-strand conformation polymorphism analysis, which is described next.
It is desirable to perform this analysis using a single gel in combination with (b) single-strand conformation polymorphism analysis (SSCP analysis; single strand
trand conformation polymorphism anal
(ysis) The amplified DNA prepared by the method described in (a) was denatured and then purified by native polyacrylamide gel electrophoresis.
When DNA amplification is performed, the primers are radioisotopes or
Amplification products can be detected by labeling with fluorescent dyes or by silver staining of unlabeled amplification products.
The amplified DNA can be detected as a band.
To clarify the difference, a control sample was also electrophoresed at the same time.
The resulting fragments can be detected by their mobility. (c) Chemical Mismatch Cleavage Method (CCM Method) Using sample-derived DNA or sample-derived cDNA as a template, the fragments of SEQ ID NO:2
By performing PCR using primers designed based on the base sequence described in
Then, the DNA encoding the polypeptide of the present invention is amplified.
A is amplified DNA containing a radioisotope or fluorescent dye.
Hybridization is then performed and mismatches are removed by treatment with osmium tetroxide.
The CCM method is the most
This is one of the most sensitive detection methods and can be applied to samples up to kilobases in length. (d) Enzymatic cleavage of mismatches Instead of osmium tetroxide as in (c) above, T4 phage resolvase and endonuclease are used.
Enzymes involved in mismatch repair in cells, such as nuclease VII, and RNA
When combined with SeA, mismatches can be enzymatically cleaved. (e) Denaturing gradient gel electrophoresis
(DGGE method) The amplified DNA prepared by the method described in (c) is subjected to a chemical denaturant gradient or temperature gradient.
The amplified DNA fragments are electrophoresed using a gel with a gradient.
It moves to the position where it is denatured into a strand, and then stops moving after denaturation. There is a mutation in the DNA.
The mobility of the amplified DNA in the gel differs between the cases where the mutation was present and where it was not present.
To increase the detection sensitivity, it is possible to detect the presence of each primer.
It is recommended to add a poly(G:C) tail to the DNA. Another method for detecting mutations in the DNA is the protein truncation test.
in truncation test: PTT method) [Genomics,20 , 1-4 (1994)].
Specific for baseshift mutations, splice site mutations, and nonsense mutations
Specifically, the cDNA derived from the specimen (which is inserted into the chromosomal gene) can be detected.
If no DNA is available, DNA derived from the specimen can also be used) as a template.
PCR is carried out using primers designed based on the base sequence described in No. 2.
The DNA encoding the full-length polypeptide of the present invention is amplified by the
The 5' end of the primer corresponding to the N-terminal side of the polypeptide contains a T7 promoter
The amplified DNA is then amplified using a sequence containing a nucleotide sequence and a eukaryotic translation initiation sequence.in v
itroA polypeptide can be produced by performing transcription and translation.
When the polypeptide is electrophoresed on a gel, the polypeptide is deleted from its permanent position.
If the electrophoretic position of the polypeptide is different from that of the full-length polypeptide, it is possible to know whether or not there is a mutation that occurs.
If the position of the polypeptide corresponds to the position of the polypeptide, no mutation will result in a deletion.
If the peptide has a deletion, the deletion is located at a position smaller than the full-length polypeptide.
The resulting DNA is electrophoresed, and the location of the mutation can be predicted from the resulting position. For mutations outside the coding region of the chromosomal gene encoding the polypeptide of the present invention,
In patients with diseases caused by mutations in non-coding regions,
Abnormalities in mRNA size and expression level were detected, so Northern hybridization
These abnormalities can be examined by PCR or PCR. If the presence of a mutation in a non-coding region is suggested,
The presence of mutations in the transcriptional regulatory region, intron region, or
The gene region is obtained by hybridizing DNA having the base sequence shown in SEQ ID NO: 2.
By using it as a probe for DNA redox amplification, it can be cloned.
In addition, the sequence information of the above gene region can be obtained from human genome sequences registered in various databases.
It may also be obtained by comparing with the human chromosomal gene sequence.
As shown in Figure 1, in the case of a chromosomal gene encoding a polypeptide of the present invention,
Mutations in non-coding regions were detected by either of the methods described above.
The DNA polymorphism analysis of the present invention can be carried out using the gene sequence information of the DNA.
Specifically, Southern blotting, direct sequencing, and PCR are possible.
Gene polymorphisms can be analyzed using methods such as DNA chips [clinical tests,
42, 1507-1517 (1998), Clinical Examination,42, 1565-1570
(1998)]. The mutations and polymorphisms found are listed in the Handbook of Human Genes
tics Linkage. The John Hopkins University
City Press, Baltimore (1994)
By performing statistical processing, SNPs (single nucleotide polymorphisms) that are linked to diseases can be identified.
In addition, the history of the above diseases can be identified as
By obtaining DNA from family members who have the disease and detecting the mutation using the method described above,
Disease diagnosis can be performed. (iv) Inhibition of transcription and translation of DNA encoding the polypeptide of the present invention The DNA of the present invention can be used in antisense RNA/DNA technology [Bioscience and Informatics].
Industry,50, 322 (1992), Chemistry,46, 681 (1991),
Biotechnology,9, 358 (1992), Trends in
Biotechnology,10, 87 (1992), Trends in
Biotechnology,10, 152 (1992), Cell Engineering,16, 1
463 (1997)], triple helix technology [Trends in Biology
otechnology,10, 132 (1992)], etc., and
The transcription or translation of DNA encoding a protein can be inhibited. For example,
By administering the DNA or oligonucleotide of the present invention,
In other words, the DNA or oligonucleotide of the present invention can inhibit the production of the peptide.
The polypeptide of the present invention can be encoded by using a nucleotide or a derivative thereof.
transcription of the DNA encoding the polypeptide of the present invention, or translation of the mRNA encoding the polypeptide of the present invention.
The method for suppressing the above-mentioned diseases can be used in the present invention to suppress inflammatory diseases, cancer, cancer metastasis, etc.
For the treatment or prevention of diseases accompanied by altered expression of DNA encoding the polypeptide
The polypeptides of the present invention can be used as medicines for the treatment of cancer. The polypeptides of the present invention inhibit the activity of neolactoglycolipids and lactoglycolipids in cancer cells.
Therefore, the transcription and polynucleotide synthesis of the DNA of the present invention are
Cancer treatment is possible by inhibiting the translation of mRNA encoding the polypeptide
It is thought that, considering the above-mentioned mechanisms of inflammatory response and cancer metastasis, polyclonal antibodies on leukocytes and cancer cells
-Inhibition of inflammatory responses by suppressing the expression of N-acetyllactosamine sugar chains
It is expected that the transcription and polyclonalization of the DNA of the present invention will be effective in suppressing cancer metastasis.
By inhibiting the translation of mRNA that encodes the peptide, it is possible to
It is possible to suppress the expression of poly-N-acetyllactosamine sugar chains in
In addition, the formation of poly-N-acetyllactosamine sugar chains in certain white blood cells and cancer cells has been shown to be
The Galβ1,3-N-acetylglucosaminyltransferases involved in the synthesis are different.
Lactosylceramide β1,3-N-acetylglucosamine
Transferases bind to poly-N-acetyllactosamine sugars in certain white blood cells and cancer cells.
It may be involved in the synthesis of the DNA of the present invention and the synthesis of the polypeptide.
The polypeptide is expressed by suppressing the translation of mRNA encoding the polypeptide.
The synthesis of poly-N-acetyllactosamine sugar chains in specific leukocytes and cancer cells is
It is believed that this can differentially suppress the expression of the DNA or oligonucleotide of the present invention. (v) Pharmaceuticals containing the DNA or oligonucleotide of the present invention Pharmaceuticals containing the DNA of the present invention, a partial fragment of the DNA, or an oligonucleotide of the present invention
The pharmaceutical containing the polypeptide of the present invention is similar to the pharmaceutical containing the polypeptide of the present invention described in (3).
The polypeptides of the present invention can be produced and administered by the method described above. (6) Production of antibodies that recognize the polypeptides of the present invention (i) Production of polyclonal antibodies Purified preparations of full-length or partial fragments of the polypeptides obtained by the method described above in (3)
Alternatively, a peptide having a partial amino acid sequence of the protein of the present invention may be used as an antigen.
Polyclonal antibodies can be produced by administering the antibody to animals. Rabbits, goats, rats, mice, hamsters, etc. are used as the animals to be administered.
The dose of the antigen is preferably 50 to 100 μg per animal.
When peptides are used, the peptides are ligated to keyhole limpet hemocyanin (keyhole limpet hemocyanin).
Carriers such as limpet hemocyanin and bovine thyroglobulin
It is preferable to use a peptide as an antigen that is covalently bound to a protein.
It can be synthesized using a peptide synthesizer. The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration.
After the immunization, blood is collected from the venous plexus of the eyeground on the 3rd to 7th day, and the serum reacts with the antigen used for immunization.
This is based on the enzyme immunoassay method (ELISA method), published by Igaku Shoin in 1976.
Year, Antibodies-A Laboratory Manual, Col.
Spring Harbor Laboratory (1988)
It is recognized that the serum of non-human mammals showed sufficient antibody titers against the antigen used for immunization.
The serum is then separated and purified to obtain polyclonal antibodies.
Methods for separation and purification include centrifugation and 40-50% saturated ammonium sulfate.
Salting out by caprylic acid precipitation [Antibodies, A Laboratory
y manual, Cold Spring Harbor Laborato
ry, (1988)], or DEAE-Sepharose column, anion exchange column
Chromatography using a column, protein A or G column, or gel filtration column, etc.
Examples of methods include the use of techniques such as fluoroscopy, either alone or in combination. (ii) Production of monoclonal antibodies (a) Preparation of antibody-producing cells Serum is produced by immunizing a partial fragment of the polypeptide of the present invention against the antibody-producing cells.
Rats that showed sufficient antibody titers were used as a source of antibody-producing cells. Three to seven days after the final administration of the antigenic substance to the rats that showed the antibody titers, the spleens were removed.
The spleen was minced in MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and loosened with tweezers.
After centrifugation at 1,200 rpm for 5 minutes, the supernatant was discarded.
spleen cells were treated with Tris-ammonium chloride buffer (pH 7.65) for 1-2 minutes.
After removing red blood cells, the resulting spleen cells were washed three times with MEM medium, and the resulting spleen cells were isolated as antibody-producing cells.
(b) Preparation of myeloma cells Myeloma cells are established cell lines obtained from mice or rats. For example, the 8-azaguanine-resistant mouse (BALB/c-derived) myeloma cell line P3
-X63Ag8-U1 (hereinafter abbreviated as P3-U1) [Curr. Topics.
Microbiol. Immunol. ，81, 1 (1978), Europe.
J. Immunol. ，6, 511 (1976)], SP2/0-Ag14 (S
P-2) [Nature,276, 269 (1978)], P3-X63-Ag
8653 (653) [J. Immunol. ，123, 1548 (1979)]
, P3-X63-Ag8(X63) [Nature,256, 495 (1975
) )], etc. can be used. These cell lines can be grown in 8-azaguanine medium (RPMI-1640 medium containing glutathione).
amine (1.5 mmol/L), 2-mercaptoethanol (5 × 10⁻⁵M),
Gentamycin (10 μg/ml) and fetal calf serum (FCS) (CSL)
, 10%) (hereinafter referred to as normal medium) was further treated with 8-azaguanine.
(15 μg/ml) was added to the medium.
The cells were cultured in a medium, and 2 × 10⁷(c) Hybridoma Production The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) are cultured in MEM medium or
or PBS (disodium phosphate 1.83 g, monopotassium phosphate 0.21 g,
The cells were washed thoroughly with 7.65 g of sodium chloride, 1 liter of distilled water, pH 7.2, and the number of cells was measured by antibody.
The production cells and myeloma cells were mixed at a ratio of 5 to 10:1, and the mixture was stirred at 1,200 rpm for 5 minutes.
After centrifugation for 1 minute, discard the supernatant. The cells in the resulting precipitated fraction are thoroughly loosened and the cells are stirred at 37°C.
So, 10⁸Polyethylene glycol-1000 (PEG) per antibody-producing cell
-1000) 2g, MEM 2ml and dimethyl sulfoxide (DMSO) 0.
Add 0.2 to 1 ml of the mixed solution of 7 ml of MEM medium every 1 to 2 minutes.
Add 1-2 ml several times. After the addition, add MEM medium to bring the total volume to 50 ml.
Prepare as follows. Centrifuge the prepared solution at 900 rpm for 5 minutes, then discard the supernatant.
After gently loosening the cells, loosen them by sucking and blowing them out with a measuring pipette.
Immediately add hypoxanthine to HAT medium (normal medium (10⁻⁴M), thymidine (1.
5 x 10⁻⁵M) and aminopterin (4 × 10⁻⁷Medium containing 10
Dispense 100 μl of the suspension into each well of a 96-well culture plate and incubate in 5% CO₂stomach
The cells are cultured in an incubator at 37°C for 7 to 14 days. After the culture, a portion of the culture supernatant is
Antibodies, A Laboratory
annual, Cold Spring Harbor Laboratory,
Chapter 14 (1988) and other methods.
A hybrid specifically reacting with a partial fragment of the polypeptide of the present invention.
A specific example of an enzyme immunoassay is as follows: During immunization, a partial fragment polypeptide of the polypeptide of the present invention used as an antigen is appropriately
The hybridoma culture supernatant or the hybridoma culture supernatant obtained in (d) described below is coated on a suitable plate.
The purified antibody is reacted as the first antibody, and biotin, enzyme,
Anti-rat or anti-mouse immunolabeled with chemiluminescent or radioactive compounds
After reacting with the globulin antibody, a reaction according to the labeling substance is carried out to obtain the poly(Ag) of the present invention.
The polypeptide monoclonal antibody of the present invention is a monoclonal antibody that specifically reacts with the peptide.
The hybridoma is selected as the one to produce the desired product. Cloning is repeated twice by limiting dilution using the selected hybridoma.
The first time was in HT medium (a medium in which aminopterin was removed from HAT medium), and the second time was in
A normal medium is used], and the antibody that shows a stable and strong antibody titer is used as the polyclonal antibody of the present invention.
A hybridoma line producing an anti-polypeptide antibody against the peptide was selected. (d) Preparation of monoclonal antibodies Pristane treatment [2,6,10,14-tetramethylpentadecane (Pris)]
0.5 ml of ethanol was intraperitoneally administered to 8-10 week old mice and the mice were then reared for 2 weeks.
The polypeptide monoclonal antibody of the present invention obtained in (c) was then transfected into a mouse or nude mouse.
5-20 x 10 cloned antibody-producing hybridoma cells⁶Cells/animal are injected intraperitoneally
The hybridomas develop into ascites tumors within 10 to 21 days.
The ascites fluid was collected and centrifuged at 3,000 rpm for 5 minutes to remove solids.
Monoclonal antibodies were extracted from the supernatant using the same method as used for polyclonal antibodies.
The antibody subclass can be determined using a mouse monoclonal antibody typing kit or
The protein amount is determined using a rat monoclonal antibody typing kit.
Calculations are made using the Lee method or absorbance at 280 nm. (e) Neutralizing Antibodies The antibodies of the present invention have the ability to neutralize the polypeptides of the present invention by binding to them.
The method according to (3) includes a neutralizing antibody having the property of suppressing the activity of the peptide.
When the activity of the polypeptide of the present invention is measured by the method, the antibody obtained above is added.
The activity of the polypeptide of the present invention was measured by adding the antibody.
If the activity is reduced, the antibody can be confirmed as a neutralizing antibody. (7) Use of the Antibody of the Present Invention (i) Detection and Quantification of the Polypeptide of the Present Invention An antibody that specifically recognizes the polypeptide of the present invention is used to carry out an antigen-antibody reaction.
By this, the polypeptide of the present invention or a cell or tissue containing said polypeptide can be
The tissue can be immunologically detected. The detection method can be used to detect inflammatory diseases, cancer, or
The present invention can be used to diagnose diseases accompanied by altered expression of the polypeptide of the present invention, such as metastasis of the breast cancer.
This detection method can also be used to quantify proteins. Immunological detection and quantification methods include fluorescent antibody techniques and enzyme immunoassays (
ELISA method), radioactive immunoassay (RIA), immunohistochemical staining and immunoassay
Immunohistochemical staining methods such as cell staining (ABC method, CSA method, etc.), Western blot
Blotting method, dot blotting method, immunoprecipitation method, sandwich ELISA
Method A [Monoclonal Antibody Experiment Manual (Kodansha Scientific) (198
7), Continued Biochemistry Experiment Course 5, Immunobiochemistry Research Methods (Tokyo Kagaku Dojin) (1986)
etc. The fluorescent antibody technique is a method for detecting microorganisms in which the polypeptide of the present invention is expressed intracellularly or extracellularly.
Reacting the antibody of the present invention with an organism, animal cell, or insect cell or tissue, and
was labeled with a fluorescent substance such as fluorescein isothiocyanate (FITC).
After reacting with anti-mouse IgG antibody or its fragment, a fluorescent dye was applied to the sample using a flow cytometer.
This is a method of measuring the amount of a protein in a cell using an enzyme-linked immunosorbent assay (ELISA).
The antibody of the present invention is expressed extracellularly in microorganisms, animal cells, or insect cells or tissues.
Antibody labeled with enzymes such as peroxidase and biotin
After reacting with mouse IgG antibody or binding fragment, the colored dye is measured with an absorption spectrophotometer.
Radioactive immunoassay (RIA) is a method in which the polypeptide is intracellularly or intracellularly.
The antibodies of the present invention are added to extracellularly expressed microorganisms, animal cells, or insect cells or tissues.
Anti-mouse IgG antibody or its fragment, which has been reacted with the antibody and further radiolabeled.
After reacting with the polypeptide, measurements are made using a scintillation counter or similar device. Immunocytostaining and immunohistostaining are methods in which the polypeptide is detected intracellularly or intracellularly.
The polypeptide is exogenously expressed in microbial, animal or insect cells or tissues.
The antibody that specifically recognizes the target substance is reacted with the target substance, and then fluorescent substances such as FITC and peroxidase are added.
Anti-mouse IgG antibody or its fragments labeled with enzymes such as ribozyme and biotin.
Western blotting is a method in which a fragment is reacted with a protein and then observed under a microscope. Western blotting is a method in which the polypeptide is detected inside or outside a cell.
The extract of the expressed microorganism, animal cell, or insect cell or tissue is diluted with SDS-polysaccharide.
Acrylamide gel electrophoresis [Antibodies-A Laboratory
y Manual, Cold Spring Harbor Laborato
After fractionation using a PVDF membrane or nitrocellulose membrane, the gel was
The polypeptide of the present invention is specifically recognized by blotting the antibody onto a membrane.
The antibody is reacted with a fluorescent substance such as FITC, peroxidase, or biotin.
After reacting with an anti-mouse IgG antibody or a fragment thereof labeled with an enzyme such as
This is a method of performing a reaction according to the labeling substance. The dot blotting method is a method of detecting the polypeptide expressed intracellularly or extracellularly.
Extracts of the extracted microorganisms, animal cells, or insect cells or tissues are then applied to nitrocellulose.
The antibody of the present invention is reacted with the membrane by blotting, and further, FITC or the like is added.
Anti-mouse IgG labeled with enzymes such as fluorescent substances, peroxidase, and biotin
This method involves reacting with an antibody or binding fragment, followed by a reaction according to the labeling substance.
Immunoprecipitation is a method for immunoprecipitation in which the polypeptide of the present invention is expressed intracellularly or extracellularly.
Extracts of organisms, animal cells, or insect cells or tissues are subjected to specific cleavage of the polypeptide.
After reacting with an antibody that recognizes the protein, the protein is then transferred to an immunoglobulin such as protein G-Sepharose.
This method involves adding a carrier that has specific binding ability to the antibody to precipitate the antigen-antibody complex.
The sandwich ELISA method uses an antibody that specifically recognizes the polypeptide of the present invention.
The polypeptide was then transferred to a plate adsorbed with the antibody, and the antibody was then transferred to a plate adsorbed with the antibody.
After reacting with the extract of microorganisms, animal cells, or insect cells or tissues, FIT
C, or enzymes such as peroxidase and biotin.
An antibody that specifically recognizes the polypeptide of the invention (having an antigen recognition site different from that of the above antibody)
In addition to the polypeptide of the present invention, two types of Galβ1,3-N-acetyltransferases have already been developed.
Although the glucosaminyltransferase has been cloned, the specific Galβ1,3-
To detect the expression of N-acetylglucosaminyltransferase, specific antibodies were used.
Therefore, it is necessary to use immunological detection based on the antibody of the present invention.
This will enable accurate analysis of the expression of the polypeptide. (ii) Use in the diagnosis and treatment of inflammatory diseases and cancer Changes in the expression levels of the polypeptide in human biological samples and human primary culture cells
Identifying structural changes in expressed polypeptides will be useful in the future to prevent disease development.
This is useful for understanding the risk of developing a disease and the causes of diseases that have already developed. The polypeptides of the present invention inhibit the activity of neolactoglycolipids and lactolipids in leukocytes and cancer cells.
Involved in the synthesis of glycolipids or poly-N-acetyllactosamine sugar chains
Therefore, it is possible to detect leukocytes or cancer cells using the antibody of the present invention.
The expression level of the polypeptide of the present invention in cells or the like is examined, and the neutralizing antibody of the present invention is used to
By controlling the activity of the polypeptide of the present invention in
This will enable the diagnosis, prevention, and treatment of various conditions. In addition, the expression of lactosylceramide β1,3-N-acetylglucosaminyltransferase will be improved.
This expression controls the differentiation of blood cells, the mutual recognition and migration of nerve cells.
It is thought that abnormal expression of this enzyme, decreased activity due to mutation of this enzyme, or inactivation of this enzyme are also thought to be involved.
Therefore, the antibody of the present invention can be used to treat various diseases.
, examining the expression level of the polypeptide of the present invention in blood cells or nerve cells,
By controlling the activity of the polypeptide of the present invention using the neutralizing antibody of the present invention,
Diseases involving the differentiation of blood cells or the mutual recognition and migration of nerve cells
This will enable the diagnosis, prevention, and treatment of myeloid leukemia and lymphocytic leukemia. For example, the treatment for these two types is different.
If there were a method to accurately distinguish between these two types of leukemia, it would be extremely useful in medical practice.
In myeloid cell lines, lactosylceramide β1,3-N-acetylglucosamine
Transferase activity is detected in lymphoid cell lines, but not in lymphoid cell lines
Therefore, the polypeptide of the present invention is expressed in myeloid leukemia cells, and lymphocytic leukemia
It is presumed that the polypeptide of the present invention is not expressed in leukemia cells.
The expression of this enzyme protein in leukemia cells collected from the
This may enable differentiation of myeloid leukemia from lymphocytic leukemia. In addition, the presence of poly-N-acetyllactosamine sugar chains in certain leukocytes and cancer cells may be
The Galβ1,3-N-acetylglucosaminyltransferases involved in the synthesis are different.
Lactosylceramide β1,3-N-acetylglucosamine
Transferases bind to poly-N-acetyllactosamine sugars in certain white blood cells and cancer cells.
It is possible that the polypeptide of the present invention is involved in the synthesis of lactosylation chains.
Gluceramide β1,3-N-acetylglucosaminyltransferase activity was determined by neutralizing antibodies
By suppressing the expression of the polypeptide, the polypeptide of specific white blood cells or cancer cells can be suppressed.
The synthesis of N-acetyllactosamine sugar chains can be specifically inhibited. Methods for detecting and diagnosing the expression level or structural changes of the polypeptide include the above-mentioned
Fluorescent antibody method, enzyme-linked immunosorbent assay (ELISA), radioactive substance-labeled immunosorbent assay (R
IA), immunohistochemical staining methods such as immunohistochemical staining and immunocytochemical staining (ABC method)
, CSA method, etc.), Western blotting method, dot blotting method, immunoassay
Examples of methods for diagnosing diseases using the above methods include precipitation methods and sandwich ELISA methods. Samples used for diagnosis using the above methods include inflammatory diseases, cancer, cancer metastasis, etc.
The polypeptide of the present invention is obtained from a patient with a disease known to be accompanied by an altered expression level.
The obtained biological sample itself, such as tissue, blood, serum, urine, feces, saliva, etc., or the biological sample itself
Cells and cell extracts obtained from samples are used.
The tissue obtained was isolated as paraffin or cryostat sections and used.
Immunological detection methods include ELISA using microtiter plates.
Examples of immunological quantification methods include SA method, fluorescent antibody method, Western blotting method, and immunohistochemical staining method. Immunological quantification methods include those using a compound that reacts with the polypeptide of the present invention in a liquid phase.
A sandwich antibody was prepared using two types of monoclonal antibodies with different epitopes.
Chi ELISA method,¹²⁵The polypeptide of the present invention is labeled with a radioisotope such as I.
Examples of such methods include radioimmunoassays using an antibody that recognizes the polypeptide of the present invention.
The pharmaceutical containing the antibody of the present invention contains the polypeptide of the present invention described in (3).
The polypeptides of the present invention can be produced and administered in a manner similar to that of pharmaceuticals containing the polypeptides of the present invention. (8) Method for preparing a recombinant viral vector that produces the polypeptides of the present invention Below is a description of a recombinant viral vector for producing the polypeptides of the present invention in specific human tissues.
This section describes a method for preparing a virus vector. Based on the full-length cDNA of the DNA of the present invention, the polypeptide can be expressed as needed.
A DNA fragment of an appropriate length containing the coding portion is prepared. For example, SEQ ID NO: 2
DNA having a base sequence represented by SEQ ID NO: 2,
DNA having the 1268th base sequence, or the base sequence represented by SEQ ID NO: 2
Examples of such DNA include DNA having the 249th to 1268th base sequence of the full-length cDNA or a DNA fragment thereof, which is inserted into a promoter in a viral vector.
A recombinant viral vector is constructed by inserting a gene homologous to the full-length cDNA of the gene of the present invention downstream of the gene.
cRNA or DNA of an appropriate length containing the portion encoding the polypeptide
RNA fragments homologous to the A fragment are prepared and used as promoters in viral vectors.
The RNA fragment is inserted downstream of the target to create a recombinant virus.
In addition to double-stranded, sense or antisense strands are available depending on the type of viral vector.
Select one of the strands. For example, in the case of a retroviral vector,
In the case of Sendai virus vectors, the RNA homologous to the sense strand is
RNA homologous to the antisense strand is selected. The recombinant viral vector is then introduced into packaging cells compatible with the vector.
The packaging cells are those that are defective in the corresponding recombinant viral vector.
It complements proteins encoded by genes required for packaging of the virus it contains.
Any cells that can supply the following proteins can be used.
It is possible to use HEK293 cells derived from liver or mouse fibroblast cells NIH3T3.
Yes, it is possible. The proteins supplied to packaging cells include retroviral vectors.
In the case of mouse retroviruses, proteins such as gag, pol, and env are used.
However, in the case of lentiviral vectors, gag, pol, e derived from HIV virus are used.
Proteins such as nv, vpr, vpu, vif, tat, rev, and nef,
In the case of adenovirus vectors, proteins such as E1A and E1B derived from adenovirus are used.
In the case of adeno-associated virus, proteins are Rep (p5, p19, p40), Vp
In the case of Sendai virus, proteins such as NP, P/C, L,
Examples of viral vectors include proteins such as M, F, and HN. Recombinant viruses are produced in the packaging cells described above.
and contains a promoter in a position where it can transcribe the DNA of the present invention in the target cell.
As a plasmid vector, MFG [Proc. Na
tl. Acad. Sci. USA,92, 6733-6737 (1995)],
pBabePuro [Nucleic Acids Res. ，18, 3587
-3596 (1990)], LL-CG, CL-CG, CS-CG, CLG [J
our own of Virology,72, 8150-8157 (1998
)], pAdex1 [Nucleic Acids Res. ，23, 3816
-3821 (1995)], etc. are used. Any promoter that can be expressed in human tissues can be used.
For example, immediate endothelial cell death (IE) of cytomegalovirus (human CMV) can be
the promoter of the early gene, the early promoter of SV40,
Retroviral promoter, metallothionein promoter, heat shock
protein promoter, SRα promoter, etc.
Alternatively, the enhancer of the IE gene of human CMV may be used together with the promoter. Methods for introducing a recombinant viral vector into packaging cells include, for example,
Calcium phosphate method [Japanese Patent Laid-Open Publication No. 2-227075], lipofection method [
Proc. Natl. Acad. Sci. USA,84, 7413 (1987)
] etc. Viruses containing the DNA of the present invention and RNA consisting of a sequence homologous to said DNA
The vector is used as a gene therapy agent to treat inflammatory diseases, cancer, cancer metastasis, etc.
Accompanying changes in the expression of the polypeptide of the present invention and the DNA encoding said polypeptide
The polypeptides of the present invention can be used as pharmaceuticals for the treatment or prevention of diseases. (9) Application to Screening Methods The polypeptides of the present invention can be used to detect β1,3-N-acetylglucosaminyltransferase activity.
That is, N-acetyltransferase binds to the galactose residue at the non-reducing end of the sugar chain via a β1,3 bond.
Specifically, i) galactose has the activity of transferring galactose.
, N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3
GlcNAc or lactose (Galβ1-4Glc), ii) galactose
, N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure
and iii) an oligosaccharide having a structure at the non-reducing end, and iii) lactosylceramide or
Galactose, N-acetyllactosamine, Galβ, including lagloboside, etc.
A complex carbohydrate having a 1-3GlcNAc or lactose structure at the non-reducing end is selected.
The β1,3 bond is formed between the galactose residue at the non-reducing end of the sugar chain of the acceptor substrate.
Therefore, the present invention has the activity of transferring N-acetylglucosamine.
A compound that changes the above activity by contacting the polypeptide with a test sample.
The polypeptides of the present invention can be used to screen for compounds that inhibit the activity of neolactoglycolipids and lactolipids in various cells.
Involved in the synthesis of glycolipids or poly-N-acetyllactosamine sugar chains
Therefore, the β1,3-N-acetylglucosaminyltransferase of the polypeptide
The activity of neolacto-series glycolipids in cells was enhanced or inhibited by a compound.
Increases the synthesis of lactoglycolipids or poly-N-acetyllactosamine sugar chains
or can be reduced. Furthermore, the transcription process of the gene encoding the polypeptide or the transcription product
Compounds that promote or inhibit the translation process into a protein can be used to inhibit the expression of the polypeptide.
and regulates the production of neolactoglycolipids, lactoglycolipids, or poly-
It is possible to control the amount of N-acetyllactosamine sugar chains synthesized. The amount of sialyl Lewis x sugar chains and sialyl Lewis x sugar chains present on poly-N-acetyllactosamine sugar chains can be controlled.
The aryl Lewis a sugar chain is known to be a ligand for selectins.
Therefore, compounds that suppress the synthesis of poly-N-acetyllactosamine sugar chains have anti-inflammatory properties.
On the other hand, poly-N-acetyllactosamine sugar chains
Compounds that increase the amount of synthesis of poly-N-acetyllactosamine sugar chains
It is thought to be useful for producing glycoconjugates with poly-N-acetyllactosamine sugar chains.
The compound can be obtained by the methods (i) to (vi) below. (i) The polypeptide of the present invention (purified) prepared using the method described in (3) above.
or a cell extract or culture supernatant of a transformant expressing said polypeptide.
) as an enzyme, and in the presence of a test compound, β1
β1,3-N-acetylglucosaminyltransferase activity was measured, and
Select compounds that increase or decrease glucosaminyltransferase activity
(ii) Cells expressing the polypeptide of the present invention or the polypeptides described in (2) above are obtained.
The transformants are cultured in the presence of the test compound for 2 hours to 1 week by the culture method described in (2) above.
After culturing for 10 min, paragloboside or poly-N-acetyllactosamine sugars on the cell surface were removed.
The amount of chain was measured using an antibody that recognizes paragloboside or poly-N-acetyllactosine.
Antibodies that recognize aminoglycosides (anti-i antibodies, anti-I antibodies) and lectins (LEA, PWM,
A compound having the activity of increasing or decreasing the amount of said sugar chain as measured using DSA.
As a measurement method using the above antibodies or lectins, for example, microtiter plate
ELISA using ELISA, fluorescent antibody method, Western blotting, immunohistochemistry
Furthermore, measurement using FACS can also be used.
(iii) A large number of peptides constituting a part of the polypeptide can be bound to a plastic pipette.
The peptides are synthesized at high density on a polysaccharide or some kind of solid support and selectively bind to the peptide.
After efficient screening of compounds (WO84/03564),
), (ii) to increase β1,3-N-acetylglucosaminyltransferase activity
or reducing activity, or paragloboside or poly-N-acetyllactosamine
(iv) Cells expressing the polypeptide of the present invention are cultured in the presence of a test compound, and a compound having the activity of increasing or decreasing the amount of glycan-binding domains is selected and isolated.
After culturing for 2 hours to 1 week by the culturing method described in (2), the amount of the polypeptide in the cells was
The amount of the polypeptide is increased by measuring the amount of the antibody of the present invention described in (6) above.
A compound having the activity of increasing or decreasing the expression of the polypeptide is selected and obtained. The increase or decrease in the expression of the polypeptide can be measured using the fluorescent antibody method or enzyme immunoassay described above in (7).
Measurement method (ELISA method), radiolabeled immunoantibody assay (RIA), immunohistological staining
immunohistochemical staining methods such as immunocytochemical staining (ABC method, CSA method, etc.),
Stan blotting, dot blotting, immunoprecipitation, sandwich
It can be detected by the ELISA method. (v) Cells expressing the polypeptide of the present invention are cultured in the presence of a test compound using the above (
After culturing for 2 hours to 1 week by the culturing method described in 2), the polypeptide in the cells is coated.
The amount of DNA transcripts was determined by Northern hybridization as described in (4) above.
Measurement is performed using a method such as the PCR method or RNase protection assay,
A compound having the activity of increasing or decreasing the amount of the transcription product is selected and obtained. (vi) A reporter gene is linked downstream of the promoter obtained in (4) above.
A plasmid incorporating the DNA was prepared by a known method, and the plasmid was transformed into the vector described in (2) above.
and then introducing the vector into animal cells in accordance with the method described in (2) above to obtain transformants.
The transformant is cultured in the presence of a test compound for 2 to 3 hours by the culture method described in (2) above.
After one week of culture, the expression level of the reporter gene in the cells was measured by a known method [
Science Research Institute, Cancer Research Department, New Cell Engineering Experimental Protocols, Shujunsha (1993)
,Biotechniques,20, 914 (1996), J. Antibi
otics,49, 453 (1996), Trends in Biochem.
ical Sciences,20, 448 (1995), Cell Engineering,16, 5
81 (1997)], and the activity of increasing or decreasing the expression level was measured.
A compound that binds to the reporter gene is selected and obtained. For example, chloramphenicol acetyltransferase (ATP) is used as a reporter gene.
spherase (CAT) gene, β-galactosidase gene, β-lactamase
The gene, luciferase gene, green fluorescent protein (
Examples of such genes include neolactoglycolipids and lactoglycolipids in cancer cells.
Therefore, the above screening was performed to identify the
The compound that inhibits the expression of the polypeptide or DNA of the present invention can be used.
This suppresses the synthesis of neolactoglycolipids and lactoglycolipids in cancer cells.
It is believed that cancer treatment is possible. Furthermore, considering the mechanisms of inflammatory reactions and cancer metastasis described above,
-Inhibition of inflammatory responses by suppressing the expression of N-acetyllactosamine sugar chains
It is expected that the above screening will suppress the progression of cancer and prevent cancer metastasis.
The present invention also provides a method for the preparation of a polypeptide or DNA of the present invention, comprising the steps of:
This allows the expression of poly-N-acetyllactosamine sugar chains in leukocytes and cancer cells to be
It is thought that this will enable the suppression of inflammatory reactions and the prevention of cancer metastasis.
. Involved in the synthesis of poly-N-acetyllactosamine sugar chains in certain leukocytes and cancer cells
The Galβ1,3-N-acetylglucosaminyltransferases involved are different.
Lactosylceramide β1,3-N-acetylglucosaminyltransferase
The enzyme is synthesized as poly-N-acetyllactosamine sugar chains in specific leukocytes and cancer cells.
It is possible that the expression of the DNA or polypeptide of the present invention is suppressed.
The compound inhibiting the expression of the polypeptide in specific white blood cells or cancer cells expressing the polypeptide.
It is expected to specifically inhibit the synthesis of N-acetyllactosamine sugar chains.
(10) Creation of non-human knockout animals A vector containing the DNA of the present invention can be used to create a target animal, such as a cow or sheep.
Embryonic stem cells of animals such as goats, pigs, horses, chickens, and mice
A D encoding the polypeptide of the present invention on a chromosome in a stem cell
NA can be obtained by a known homologous recombination technique [e.g., Nature,326, 6110, 2
95 (1987), Cell,51, 3, 503 (1987), etc.
Alternatively, a mutant clone can be prepared by substituting any sequence [for example, N
ture,350, 6315, 243 (1991)]. The embryonic stem cell clones thus created were used to develop blastocysts (
The embryonic chimera method is performed by injection into the blastocyst or aggregation chimera method.
It is possible to create chimeric individuals consisting of stem cell clones and normal cells.
By crossing the mutant individual with a normal individual, the polynucleotides of the present invention on the chromosomes of cells in the whole body are
It is possible to obtain individuals having any mutation in the DNA encoding the peptide, and
When the individual is crossed with another individual, mutations occur in both homologous chromosomes, resulting in a homozygous individual (non-
In this way, the polypeptide of the present invention can be encoded on the chromosome of an individual animal.
For example, a mutation can be introduced into any position of the DNA of the present invention on a chromosome.
Modifications such as base substitutions, deletions, and insertions in the translation region of DNA encoding a polypeptide
By introducing mutations into the expression control region, the activity of the product can be altered. Introduction of similar mutations into the expression control region can also affect the level, timing, and tissue expression.
It is also possible to modify the specificity, etc. Furthermore, combination with the Cre-loxP system
This makes it possible to more actively control the expression time, expression site, expression amount, etc.
This method involves using a promoter that is expressed in a specific region of the brain.
, a method for deleting a target gene only in that region [Cell,87, 7, 1317
(1996)] or Cre-expressing adenovirus,
A method for organ-specific deletion of a target gene [Science,278, 5335,
(1997)]. Therefore, the DNA encoding the polypeptide of the present invention on the chromosome can also be
It is possible to control expression at any time and in any tissue, or to make any insertion, deletion, or substitution.
It is possible to create animals that contain the polynucleotides of the present invention in their translation regions or expression control regions. Such animals can express the polynucleotides of the present invention at any time, to any extent, or in any part of the body.
The present invention can induce various disease symptoms caused by peptides.
The non-human knockout animals of the present invention can be used to treat various diseases caused by the polypeptides of the present invention.
This will be an extremely useful animal model for the treatment and prevention of
It is extremely useful as a model for evaluating functional foods, health foods, etc. (11) A method for producing a nucleic acid encoding a nucleic acid comprising the DNA of the present invention and RNA having a sequence homologous to the DNA.
Gene Therapy Agent A virus containing the DNA of the present invention and RNA consisting of a sequence homologous to the DNA.
The gene therapy agent using the vector is a recombinant virus vector prepared in (7).
and a base used in gene therapy agents.
[Nature Genet. ，8, 42 (1994)].
and a viral vector containing RNA having a sequence homologous to said DNA.
The gene therapy agent is a compound comprising the polypeptide of the present invention and the polypeptide of the present invention.
for the treatment or prevention of diseases associated with altered expression of DNA encoding a polypeptide
It can be used as a medicine. As a base for gene therapy agents, any base normally used for injections can be used.
The solution may be distilled water, sodium chloride, or a mixture of sodium chloride and an inorganic salt.
Salt solutions such as mixtures, mannitol, lactose, dextran, glucose, etc.
Sugar solution, amino acid solution such as glycine or arginine, organic acid solution or salt solution
In addition, the solution may be a mixture of the above bases according to the usual method.
Pressure adjuster, pH adjuster, vegetable oil such as sesame oil or soybean oil, or lecithin or non-isopropyl alcohol
Using auxiliary agents such as surfactants, such as ionic surfactants, as solutions, suspensions, and dispersions
Injectable preparations may be prepared. These injections may be prepared by powdering, freeze-drying, etc.
The gene therapy agent of the present invention can also be prepared as a liquid preparation to be dissolved when used.
In the case of an individual, the gene is added to the above base, which has been sterilized if necessary.
It can be dissolved immediately before treatment and used for treatment.
The administration method may be a topical administration method so that the drug is absorbed into the treatment area of the patient.
Examples include the DNA of the present invention of an appropriate size fused to the adenovirus hexon protein.
Combining with specific polylysine-conjugated antibodies to form complexes
The resulting complex is then bound to an adenovirus vector.
A viral vector can be prepared by stably targeting the target gene.
It reaches the cell, is taken up into the cell by endosome, and is degraded within the cell.
In addition to retroviral vectors, RNA viral vectors include (-) strand RNA
A viral vector based on the Sendai virus, an A virus, has also been developed.
(Patent Application No. 9-517213, Patent Application No. 9-517214), aiming at gene therapy.
and preparing a Sendai virus vector incorporating the DNA of the present invention as a target.
This DNA can also be delivered to the lesion by non-viral gene transfer methods.
Non-viral gene transfer methods known in the art include calcium phosphate co-precipitation [V
irology,52, 456-467 (1973); Science,209 , 1414-1422 (1980)], microinjection method [Proc
．． Natl. Acad. Sci. USA,77, 5399-5403 (1980
); Proc. Natl. Acad. Sci. USA,77, 7380-738
4 (1980); Cell,27, 223-231 (1981); Nature
,294, 92-94 (1981)], liposome-mediated membrane fusion-mediated transfer
[Proc. Natl. Acad. Sci. USA,84, 7413-7417
(1987);Biochemistry,28, 9508-9514 (198
9);J. Biol. Chem. ，264, 12126-12129 (1989
);Hum. Gene Ther. ，3, 267-275 (1992); Sci.
ence,249, 1285-1288 (1990); Circulation
,83, 2007-2011 (1992)] or direct DNA uptake and
Receptor-mediated DNA transfer [Science,247, 1465-1468 (1
990); J. Biol. Chem. ，266, 14338-14342 (19
91); Proc. Natl. Acad. Sci. USA,87, 3655-3
659 (1991); J. Biol. Chem. ，264, 16985-169
87 (1989); BioTechniques,11, 474-485 (19
91); Proc. Natl. Acad. Sci. USA,87, 3410-3
414 (1990); Proc. Natl. Acad. Sci. USA,88,
4255-4259 (1991); Proc. Natl. Acad. Sci. U
S.A.,87, 4033-4037 (1990); Proc. Natl. Acad
. Sci. USA,88, 8850-8854 (1991); Hum. Gene
Ther.,3, 147-154 (1991)]. In the liposome-mediated membrane fusion-mediated transfer method, a liposome preparation is used as a target.
By administering the gene directly to the tissue, local uptake and expression of the gene in the tissue can be achieved.
It has been reported in tumor studies that this is possible [Hum. Gen.
e Ther.,3, 399-410 (1992)]. Therefore, the same effect can be obtained by the present invention.
It is also expected to be effective in disease lesions involving DNA and polypeptides.
For direct targeting of the foci, direct DNA uptake techniques are preferred.
Protein-mediated DNA transfer involves the attachment of DNA to a protein ligand, e.g., via polylysine.
NA (usually in the form of a covalently closed, supercoiled plasmid)
The ligand binds to the cell surface of the target cell or tissue.
The ligand-DN is selected based on the presence of the corresponding ligand receptor on the surface.
The A conjugate can be injected directly into the blood vessels if desired, and the receptor binding
and can be directed to target tissues where internalization of the DNA-protein complex occurs.
To prevent intracellular DNA destruction, adenovirus is co-infected.
It can also disrupt endosomal function. The DNA of the present invention, RNA having a sequence homologous to the DNA, or a composition containing the nucleic acid
The gene therapy agent containing the recombinant vector is introduced into cells, and the DNA is transfected.
By expressing the polypeptides encoded by the polypeptides, differentiation, mutual recognition, and migration of the cells can be promoted.
The cells that can be used in this case include blood cells, nerve cells, etc.
Examples of such gene therapy agents include promyelocytic cells, stem cells, and cancer cells. Furthermore, by introducing the above-mentioned gene therapy agent into promyelocytic cells and expressing it,
It is possible to promote the differentiation of promyelocytic cells into granulocytic cells. The present invention will be explained in detail below using examples. However, these examples
It is for illustrative purposes only and is not intended to limit the scope of the present invention.Best Mode for Carrying Out the Invention Unless otherwise specified, the genetic engineering techniques described in the following examples are based on molecular
The known method described in "Cloning," 2nd edition, was used. Example 1: β1,3-galactose from a rat tibia cDNA library
Cloning of the rat G6 transferase homolog gene (1) RNA preparation RNA was isolated from rat tibia using the guanidine thiocyanate-cesium trifluoroacetate method [M
methods in Enzymology,154, 3 (1987)]
Then, 2.2 mg of total RNA was prepared. Then, 2.0 mg of total RNA was subjected to oligo dT-PCR.
Passed through a cellulose column (Collaborative Research)
By doing so, poly(A)⁺15.7 μg of mRNA was obtained as RNA.
(2) Preparation of a cDNA library Using 4.0 μg of the mRNA obtained in (2) above, a cDNA library was prepared using the linker primer method [
Nojima Hiroshi, ed., Gene Library Construction Method, Yodosha, 1994],
A synthesis,BamAddition of HI adapter,NotThe cleavage reaction was carried out with I.
The double-stranded cDNA was cloned into the plasmid pBluescript II SK(-)
ofBamHI-NotBy inserting it between the Λ and Λ, the 5' end of the cDNA is always in the vector.
Tar'sBamA cDNA library was constructed on the HI site side.
As a host for library construction, Escherichia coli MC1061A [Molecular Cloning
Random sequencing (2nd edition) was used. (3) Random sequencing Plasmid DNA was extracted from each E. coli clone obtained in (2) above using standard methods.
The cDNA fragments were obtained by cleaving 300 to 300 nucleotides from the 5' and 3' ends of the cDNAs contained in each plasmid.
The 400 bp base sequence was determined. The base sequence was determined using a commercially available kit (Dye
Terminator Cycle Sequencing FS Ready
Reaction Kit, dRhodamine Terminator
Cycle Sequencing FS Ready Reaction K
it or BigDye Terminator Cycle Sequence
ing FS Ready Reaction Kit, PE Biosyst
ems) and a DNA sequencer (ABI PRISM 377, PE B
The primers used were T3 primer,
- (STRATAGENE) and T7 primer (STRATAGENE
(manufactured by the company) was used. The obtained base sequences were analyzed using BLAST [J. Mol. Biol.215, 4
03-410 (1990)], and the amino acid sequence predicted from the base sequence
Using the FrameSearch program (Compugen),
The homologous genes and proteins were analyzed.
The cDNA contained in the plasmid encodes β1,3-galactosyltransferase β3
A protein having homology to Gal-T1 (also known as WM1: JP-A-6-181759)
The base sequence of the cDNA contained in OVX2-038 (
738 bp) is set forth in SEQ ID NO: 3, and the polypeptide that is believed to be encoded by said DNA
The amino acid sequence of OVX2-038 is shown in SEQ ID NO: 32.
The rat β1,3-galactosyltransferase homolog (named rat G6) was used.
It was thought to be a partial fragment of the cDNA encoding human G6. Example 2: Search for the gene encoding human G6 A search was conducted to find the human G6 gene corresponding to the rat G6 cDNA obtained in Example 1.
From the gene database, BLAST [J. Mol. Biol.215,
403-410 (1990)] and FrameSearch (Compuge
Using the program of the OVX2-038 obtained in Example 1,
a gene having homology to the base sequence of the cDNA (described in SEQ ID NO: 3)
The polypeptide (described in SEQ ID NO: 32) that the cDNA is believed to encode is
Search for genes that may encode polypeptides with homology at the acid level
As a result, one EST (expressed sequence tag) sequence is generated.
The EST sequence was found to be 428
bp, and the sequence information alone does not reveal the sequence encoded by the gene corresponding to this EST.
The entire amino acid sequence of the polypeptide and its function are unknown. Example 3: Cloning of human G6 cDNA Specific to the EST sequence (GenBank number AI039637) found in Example 2,
We designed a unique primer set and attempted to clone candidate gene fragments.
The timers used were CB-462 having the base sequence represented by SEQ ID NO: 4 and
The primer set used was CB-464 having the base sequence shown in No. 5.
Single-stranded cDNA prepared from various organs or cells, or various
PCR was performed using the cDNA library as a template to check for the presence of human G6 cDNA.
As a result, a cDNA library derived from the human colon cancer cell line Colo205 was
or about 250 b when a cDNA library derived from human gastric mucosa was used as a template.
The DNA fragment of p was amplified. The amplified fragment was used as a probe to screen the two cDNA libraries.
By performing the above-mentioned PCR, a human G6 cDNA clone of approximately 3 kb was obtained.
Since the 5' end was deleted, the 5' end of the human G6 cDNA was isolated using the 5' RACE method.
The terminal DNA was obtained. A cDNA clone of approximately 3 kb of human G6 cDNA was
By determining the base sequence of the 5' end of the DNA and connecting these sequences,
The full-length sequence of human G6 cDNA (SEQ ID NO: 2) was determined.
PCR was performed using the primers to obtain a DNA fragment containing the entire coding region.
The sequence of the DNA fragment was determined, and the base sequence derived from PCR was used as a base pair.
It was confirmed that there were no mutations in the base sequence. Specific methods are as follows: (1) Creation of a cDNA library derived from the human colon cancer cell line Colo205 and P
Analysis by CR mRNA was extracted from the human colon cancer cell line Colo205 using the Oligo mRNA extraction kit.
tex^TM-dT30<super> (Roche) was used, and approximately 30 μg
The specific reagents and methods were described in the instructions attached to the kit.
The obtained mRNA (8 μg) was mixed with SUPERSCRIPT CHOICE
System for cDNA Synthesis Kit (GIBCO
Double-stranded cDNA was synthesized using oligo dT as a primer using a PCR primer (manufactured by BRL).
These double-stranded cDNAs were then ligated at both ends using the following method.SFII linker was added.
SFIA single-stranded DNA having the base sequence represented by SEQ ID NO: 6 that constitutes the I linker
A (11 bases) and a single-stranded DNA (8 bases) having the base sequence represented by SEQ ID NO: 7
The bases were synthesized using a 380A DNA synthesizer (Applied Biosystems).
) were used for synthesis. 50 μg of each of the synthetic single-stranded DNAs was separately diluted with 50 mmol/L Tris
HCl (pH 7.5), 10 mmol/l MgCl₂, 5 mmol / l
Dithiothreitol (hereinafter abbreviated as DTT), 0.1 mmol/l EDT
A buffer solution containing ethylenediaminetetraacetic acid (EDTA) and 1 mmol/L ATP (hereinafter
The T4 polynucleotide was dissolved in 50 μl of T4 kinase buffer (abbreviated as T4 kinase buffer).
Thirty units of kinase (Takara Shuzo) was added, and phosphorylation reaction was carried out at 37°C for 16 hours.
4 μg of 11-base synthetic DNA and 8-base synthetic DNA with phosphorylated 5' ends.
2.9 μg of the double-stranded cDNA synthesized above was added to 45 μl of T4 ligase buffer.
After dissolving in 1050 units of T4 DNA ligase, the mixture was reacted at 16°C for 16 hours.
, each of the double-stranded cDNAsSFII linker was added. The resulting reaction solution was subjected to agarose gel electrophoresis, and DNA fragments of approximately 1.5 kb or more were isolated.
The fragment was recovered. Direct expression cloning vector (Expression Cloning V
pAMo [J. Biol. Chem.,268, 22782
(1993), also known as pAMoPRC3Sc (JP 05-336963)
μg in 10 mmol/L Tris-HCl (pH 7.5), 6 mmol/L
MgCl₂, 50 mmol/l NaCl, 6 mmol/l 2-mercaptoethanol
Dissolved in 590 μl of a buffer solution consisting of ethanol (hereinafter referred to as Y-50 buffer solution).
Then 80 unitsSFII (manufactured by Takara Shuzo Co., Ltd.; hereafter, unless otherwise stated, restriction enzymes are Takara Shuzo Co., Ltd.
(Product manufactured by Shuzo Co., Ltd. was used) was added and the digestion reaction was carried out at 37°C for 16 hours. 40 units ofBamHI was added, and the digestion reaction was carried out at 37°C for 2 hours.
The reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of about 8.8 kb was recovered.
The above-preparedSFIDouble-stranded cDNA (mRNA 8μ
100 μl of T4 ligase buffer solution was dissolved in 250 μl of T4 ligase buffer solution.
Add 2 μg of a DNA fragment of about 8.8 kb and 2000 units of T4 DNA ligase.
The binding reaction was carried out at 16°C for 16 hours. After the reaction, 5 μg of transfer RNA (tRNA) was added to each reaction solution.
After ethanol precipitation, the mixture was diluted with 10 mmol/L Tris-HCl (pH 8.0) and
and 1 mmol/L EDTA (hereinafter referred to as TE buffer)
The reaction mixture was used for electroporation [Nucleic Acids
Res.,16, 6127 (1988)] to Escherichia coli LE392 strain [Moreki
The transformant was a strain of 1000 cells of the genus 'Matrix', which was transformed into approximately 1 million ampicillin-resistant cells.
A transformant carrying the gene was obtained, and a cDNA library was constructed. Furthermore, the cDNA library (E. coli) and a plasmid preparation kit were also prepared.
Plasmid Maxi Kit (QIAGEN, product number 410)
31), a plasmid containing cDNA was prepared. Using this plasmid DNA as a template, CB-4 having the base sequence represented by SEQ ID NO: 4 was cloned.
62 and CB-464 having the base sequence represented by SEQ ID NO: 5 as primers.
PCR was performed using the DNA fragment to examine the presence of human G6 cDNA.
10 ng/ml plasmid DNA, 10 mmol/l Tris-HCl
l (pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl
₂, 0.2 mmol/l dNTP, 0.001% (w/v) gelatin, 0.2
μmol/l human G6 gene-specific primers (CB-462 and CB-46
4), 1unit AmpliTaq Gold DNA polymeras
A reaction solution (50 μl) consisting of 100 μL ...
After heating for 11 minutes, the reaction consisted of 95°C for 30 seconds, 55°C for 1 minute, and 72°C for 2 minutes.
The reaction was repeated 50 times.
A DNA fragment of approximately 250 bp believed to be derived from human G6 cDNA was amplified from the POI. (2) Construction of a human gastric mucosa cDNA library A human gastric mucosa cDNA library was constructed as follows.
ly (A)⁺RNA to cDNA Synthesis System (cDNA Synthesis
cDNA was synthesized using a PCR system (GIBCO BRL), and both
At the edgeEcoRI-NotI-SalI Adapter (Super Choice
System for cDNA Synthesis; GIBCO BRL
After adding the cloning vector λZAP II (λZAP II/E
coRI/CIAP Cloning Kit (Stratagene)
EcoInserted into the RI site and Gigapack III Gold Packag
Extract (STRATAGENE)in vitr
oA cDNA library was created by packaging. The gastric mucosa cDNA library (phage library) was divided into approximately 50,000 independent clones.
After pooling by loan, phages (approximately 1 × 10⁷) as a mold
PCR was performed by heating phages (approximately 1 × 1) at 99°C for 10 minutes.
0⁷The method was the same as that described in (1) above, except that the P
As a result of CR, approximately 250b of a fragment thought to be derived from human G6 cDNA was identified from several pools.
The DNA fragment was amplified using Prep-A-Gene DNA
Purification kit (BIO RAD) was used to purify the product.
Restriction enzyme (AvaII orRsaI) The amplified fragment is digested to obtain the EST sequence
(GenBank number AI039637) (946th position of SEQ ID NO: 2)
It was confirmed that the amplified fragment contained the sequence shown in the 1196th position of the amplified fragment (251 bp).
The fragments areAvaII cleavage yields three DNA fragments of 96 bp, 80 bp, and 72 bp.
In fragments,RsaIt was digested into two DNA fragments of 176 bp and 72 bp by I cleavage.
(3) Cloning of human G6 cDNA Multiprime DNA Labeling System (Ame
(2) above was performed using a PBS (manufactured by Barsham Pharmacia Biotech).
The PCR-amplified DNA fragment of about 250 bp obtained in the above was labeled with a radioisotope.
A probe was prepared using 50 ng of the DNA fragment, random primers, and random
Geoisotope ([α−³²50 μl of the reaction solution containing [P]dCTP was incubated at 37°C.
The reaction mixture was prepared and operated according to the kit instructions.
The reaction was stopped by vortexing, and the mixture was separated using a Sephadex G-50 column.
The radioisotope-labeled probe was purified by gel filtration using a 100-kDa nucleotide sequence. Seven pools (approximately 1000 kDa in total) of the gastric mucosa cDNA library in which amplification was observed in (2) above were used.
350,000 independent clones) using radioisotope-labeled probes.
Plaque-derived DNA was transferred onto a filter (Biodyne A:P
ALL Co., Ltd.) was mixed with 5x concentration SSPE (the composition of 1x concentration SSPE is 180 ml
mol/l sodium chloride, 10 mmol/l sodium dihydrogen phosphate, 1 mm
100 ml/L EDTA (pH 7.4)], 5-fold concentrated Denhardt's solution [1
The composition of the double-concentrated Denhardt's solution is 0.02% (W/V) bovine serum albumin,
0.02% (W/V) Ficoll 400, 0.02% (W/V) polyvinylpyrrolidone
lysine], 0.5% sodium dodecyl sulfate (SDS), 20 μg/m
1. A buffer solution containing salmon sperm DNA (hereinafter referred to as hybridization buffer solution)
) and prehybridized at 65°C for 1 hour. The filter was then immersed in 25 ml of the radioisotope-labeled probe prepared above.
The plate was immersed in 10 ml of hybridization buffer containing the above material and hybridized at 65°C for 16 hours.
After that, 0.1x SSC (the composition of 1x SSC is 15 mmol/L) was added.
1/1 sodium citrate, 150 mmol/l sodium chloride (pH 7.
0)], and 0.1% SDS buffer at 42°C for 20 minutes.
The plaques were washed twice. As a result of the plaque hybridization, 11 independent hybridizing
A phage clone was obtained using a kit manufactured by STRATAGENE.
in vivoExcision was performed to convert each phage clone into a plasmid clone.
The insert cDNA of each phage clone was transformed into p
Obtaining a plasmid inserted into Bluescript SK(-)
The method was according to the kit's instructions. The 11 types of plasmids obtained above were digested with restriction enzymes.EcoCut with RI and each plus
The size of the cDNA contained in each plasmid was examined. As a result, all plasmids were approximately 3 kb.
It was revealed that the cDNA contained multiple restriction enzymes (PstI,Hind
III orBsmFrom the cleavage patterns of I), all 11 types of plasmids were identical.
One of these plasmids was designated pBS-G6s.
(4) Determination of the base sequence of the cDNA inserted into the plasmid pBS-G6s The entire base sequence of the cDNA contained in the pBS-G6s obtained in (3) above was determined using the following method.
The primers used were specific to the sequence in pBluescript SK(-) [M13
(-20) Primer and M13 Reverse Primer: TOY
The 5' and 3' sequences of the cDNA were determined using a PCR product manufactured by OBO.
A synthetic DNA specific to the determined sequence is prepared and used as a primer.
The base sequence of the cDNA was then determined.
The base sequence was determined. The base sequence was determined using a DNA sequencer model 4000L (LI-
COR) and reaction kit (Sequitherm EXCEL II^TML
ong-Read^TMDNA-sequencing kit-Lc: Air
Braun), or DNA Sequencer 377 (Perkin Elme
r company) and reaction kit (ABI Prism^TMBig Dye^TMTerm
inator Cycle Sequencing Ready Reacti
The cDNA contained in pBS-G6s is from bases 989 to 3750 of SEQ ID NO: 2.
It was revealed that the gene had the base sequence shown in Figure 1 (total base sequence 2762 bp).
From the base sequence, it was determined that the cDNA is a human β1,3-galactosyltransferase homolog (
It was thought to encode human G6, but the N-terminal polypeptide portion was deleted.
(5) Cloning of the full-length human G6 cDNA The results of (1) above indicate that human G6 transcripts are expressed in the colon cancer cell line Colo205.
Therefore, we used the total RNA of Colo205 as a template.
By performing the 5' RACE method using the DNA fragment, the 5' DNA fragment of human G6 cDNA was isolated.
The 5'-RACE method was performed using a kit (5'-RACE Systems f
or Rapid Amplification of cDNA Ends,
Version 2 (manufactured by Life Technologies)
The G6 cDNA-specific primers used were the base sequences shown in SEQ ID NOs: 8 and 9.
As a result, a DNA fragment of approximately 460 bp was amplified.
The DNA fragment was blunt-ended by a conventional method and then cloned into pBluescript.
SK(-)EcoThe vector was then integrated into the RV site of pBluescript.
Primers specific to the sequence in SK(-) [M13(-20) Primer and
and M13 Reverse primer: manufactured by TOYOBO Co., Ltd.]
The sequence of the DNA fragment was determined.
del 4000L (LI-COR) or DNA Sequencer 377 (
Peppkin Elmer) and reaction kits for each sequencer.
The DNA fragment (designated RO2-16) was used.
The DNA had the base sequence shown at position 969 from the base sequence (total base sequence: 457 bp).
The A fragment did not cover the N-terminal side of the G6 polypeptide.
The following experiment was carried out. The gastric mucosa cDNA library constructed in (2) above was used as a template, and the vector
PCR was performed using a primer specific to the human G6 cDNA and a primer specific to the human G6 cDNA.
By carrying out the above steps, the DNA fragment at the 5' end of human G6 cDNA is amplified.
Specifically, pBluescR
M13 Rivers, a primer specific to the sequence in ipt SK(-),
e primer (manufactured by TOYOBO), a human G6 cDNA specific primer;
A synthetic DNA having the base sequence shown in SEQ ID NO: 10 was used.
was carried out using 50 ng/ml of cDNA library (plasmid) as a template.
The PCR reaction was carried out under the conditions described in (1).
M13 Ri was used as a primer specific to the sequence in the script SK(-).
Verse primer (manufactured by TOYOBO), human G6 cDNA specific primer
PCR was performed using a synthetic DNA having the base sequence shown in SEQ ID NO: 9 as a primer.
The PCR reaction was carried out under the conditions described in (1) above. As a result, approximately 1 kb
The DNA fragment was amplified. The DNA fragment was blunt-ended according to standard methods and then inserted into pBluescript S
K(-)EcoThe vector was then integrated into the RV site of pBluescript S
A primer specific to the sequence in K(-) [M13(-20) Primer or
M13 Reverse primer: both manufactured by TOYOBO Co., Ltd.
The sequence of the DNA fragment was determined. A synthetic DNA specific to the determined sequence was prepared.
This was used as a primer to determine the further base sequence.
The entire base sequence of the DNA fragment was determined by repeating the above steps.
, DNA sequencer model 4000L (manufactured by LI-COR) or D
NA Sequencer 377 (Perkin Elmer), and each sequence
A reaction kit for the Sequencer was used. The DNA fragment (designated No. 19)
The base sequence shown from the 1st to the 969th positions of SEQ ID NO: 2 (total base sequence: 969 bp)
The cDNA sequence contained in pBS-G6s determined above, the sequence determined in (2) above
By ligating the sequence of the PCR fragment, the sequence of RO2-16, and the sequence of No. 19,
As a result, the nucleotide sequence of the cDNA encoding the full-length G6 polypeptide was determined.
The determined base sequence of the full-length human G6 cDNA (3750 bp) is shown in SEQ ID NO: 2.
The cDNA consists of 378 amino acids with a structure characteristic of glycosyltransferases.
This polypeptide was called G6 polypeptide.
The amino acid sequence is shown in SEQ ID NO: 1. To obtain DNA encoding the full-length G6 polypeptide, human colon cancer cells
Using the cDNA library of the strain Colo205 as a template, a cDNA specific for human G6 cDNA was
PCR was performed using different primers.
NA library (plasmid),EcoA primer containing an RI recognition sequence [
CB-497 (SEQ ID NO: 11) and CB-501 (SEQ ID NO: 12)], and P1
atinum Pfx DNA polymerase (GIBCO BRL)
50 μl of reaction solution containing Platinum Pfx DNA (product of
The polymerase kit was heated at 94°C for 2 minutes, and then
One cycle of reaction consisting of 20 seconds at 4°C, 45 seconds at 55°C, and 2 minutes at 68°C
PCR was carried out by carrying out 45 cycles of reaction.
A 3 kb DNA fragment was amplified.EcoAfter digestion with RI, pBlue
script SK(-)EcoBy inserting into the RI site, the plasmid
pBS-G6 was obtained. The sequence of the inserted DNA fragment was determined.
The NA fragment has the base sequence shown in SEQ ID NO: 2 from base 46 to base 1372.
The base sequence was determined using a DNA sequencer 377 (Per
(manufactured by Kin Elmer) and a reaction kit for the sequencer, and human G6
cDNA-specific primers were used. The DNA fragments were characterized by glycosyltransferases.
The polypeptide encoded was composed of 378 amino acids having the structure
The peptide had the same amino acid sequence as the G6 polypeptide.
By performing direct sequencing using the approximately 1.3 kb DNA fragment obtained,
Therefore, there were no errors in the sequence of the PCR-amplified DNA fragment inserted into pBS-G6.
It was confirmed that the cDNA sequence contained in pBS-G6s determined above, the sequence of No. 19, and
The sequence of the PCR-amplified DNA fragment contained in pBS-G6 was ligated to
The nucleotide sequence of the cDNA encoding the full-length human 6 polypeptide was determined.
The base sequence of the full-length G6 cDNA (3750 bp) is the same as that of SEQ ID NO: 2.
Example 4: Homology Analysis The G6 polypeptide is homologous to the five human β1,3-galactosidases that have been cloned to date.
intosyltransferase β3Gal-T1 (JP Patent Publication No. 6-181759), β3Gal-
T2 [J. Biol. Chem. ，273, 433-440 (1998), J.
Biol. Chem. ，273, 12770-12778 (1998)], β3
Gal-T3 [J. Biol. Chem. ，273, 12770-12778 (
1998)], β3Gal-T4 [J. Biol. Chem. ，273, 127
70-12778 (1998)], β3Gal-T5 [J. Biol. Chem
．274, 12499-12507 (1999)] and at the amino acid level, respectively.
35.6%, 32.8%, 30.8%, 30.3%, and 33.9% homology
Furthermore, the polypeptide was found to be similar to the previously cloned human β1,3
-N-acetylglucosaminyltransferase (β3GnT1) [Proc. Natl.
Acad. Sci. USA,96, 406-411 (1999)] and amino acid residues
The amino acid sequence (SEQ ID NO: 1) of the G6 polypeptide shows a homology of 29.4% between the G6 and G6 polypeptides, which is characteristic of glycosyltransferases.
It is thought to be a type II membrane protein.
The cytoplasmic domain is followed by a hydrophobic membrane-binding domain consisting of 18 amino acids.
a stem region of at least 12 amino acids, and most of the remaining C-terminus, which contains the catalytic domain
The amino acid sequence of the glycosyltransferase with homology
Comparison, and findings regarding the stem region and catalytic region of the above glycosyltransferase [Patent Publication No. 6-1
Based on the results of the analysis of the nucleotide sequence ...
Therefore, the polypeptide containing the amino acid sequence from 45th to 378th is a catalytic domain.
These results and the results of Examples 6, 7, 9 and 10 described below indicate that the poly
The peptide is a novel β1,3-N-acetylglucosaminyltransferase and has sequence number
36 to 378 of SEQ ID NO: 1 and 39 to 378 of SEQ ID NO: 1
It has been revealed that polypeptides containing the amino acid sequence are secreted polypeptides.
Example 5: Construction of an Expression Plasmid for Animal Cells The G6 polypeptide encoded by the human G6 cDNA obtained in Example 3 was expressed in animal cells.
For expression in cells, human G6 cDNA was introduced into the expression vector pAMo [J. Biol.
l. Chem.,268, 22782 (1993), also known as pAMoPRC3Sc
(JP-A-5-336963)], or pCXN2 [Gene,108, 19
3 (1991) to construct an expression plasmid. (1) Construction of plasmid pAMo-G6 for expressing G6 polypeptide pBS-G6 was digested with restriction enzymesEcoAfter cleavage with RI, DNA polymerase Klenow
The ends were converted to blunt ends using the fragment prepared in Example 3(1).SFII
A linker was added to form a fragment of about 1350 bp.SFIOn the other hand, pAMo
ofSFII andBamAfter digestion with HI, an 8.7 kb fragmentSFIFragment I was obtained.
The expression plasmid pAMo-G6 was constructed by ligating the two fragments. (2) Construction of the plasmid pCXN2-G6 for expressing the G6 polypeptide pBS-G6 was purified by restriction enzyme digestion.EcoAfter cleavage with RI, a fragment of approximately 1330 bp was obtained.EcoRI cutoff
On the other hand, pCXN2 wasEcoAfter cleavage with RI, a fragment of approximately 3 kb was obtained.EcoRI
The above two fragments were ligated to obtain the expression plasmid pCXN2
-G6 was constructed. Example 6: Expression of G6 polypeptide in cultured human cells transfected with an expression plasmid
Synthesis of poly-N-acetyllactosamine sugar chains (1) Obtaining stable transformants using Namalwa cells as hosts Control plasmid (pAMo) and the G6 polypeptide constructed in Example 5
The nucleotide expression plasmid (pAMo-G6) was added to the nuclease-containing nucleotide sequence at a concentration of 1 μg/μl.
After dissolving in TE buffer, the cells were subjected to electroporation [Cytotechnol
ogy,3, 133 (1990)] into the human B cell line Namalwa cells.
and transformed cells were obtained. 1.6 x 10⁶After transfection with 4 μg of plasmid per cell, 10% of bovine fetuses
RPMI 1640 medium containing fetal serum [7.5% NaHCO₃1/40 of the amount, 2
300 mmol/L L-glutamine solution (GIBCO) 3%, penicillin
Streptomycin solution (GIBCO, 5000 units/ml penicillin)
RPMI1 supplemented with 0.5% phosphate buffer (phosphate buffer, 5000 μg/ml streptomycin)
640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.). Hereinafter, RPMI 1640 medium refers to the medium containing the above-mentioned additives.
RPMI 1640 medium supplemented with 8 ml of CO₂Incubator
The cells were cultured in a hood at 37°C for 24 hours. After the culture, G418 (GIBCO) was added to
The resulting mixture was added to a concentration of 8 mg/ml and cultured for another 14 days to obtain stable transformants.
The transformant was cultured in 0.8 mg/ml G418 and 10% fetal bovine serum.
The cells were subcultured in RPMI 1640 medium containing 100% ethanol. (2) Obtaining stable transformants using HCT-15 cells as hosts The control plasmid (pCXN2) and the G6 polypeptide constructed in Example 5 were used.
The peptide expression plasmid (pCXN2-G6) was added to the 500-kDa ...
After dissolving in TE buffer, the cells were subjected to electroporation [Cytotechn
ology,3, 133 (1990)] to identify the human colon cancer cell line HCT-15 cells.
The cells were transfected with the vector to obtain transformed cells. 8 x 10⁶After transfection with 10 μg of plasmid per cell, 10% fetal bovine
The cells were suspended in 8 ml of serum-containing RPMI 1640 medium and₂Incubator 3
The cells were cultured at 7°C for 24 hours. After culturing, G418 (GIBCO) was added to a concentration of 0.8 mg/ml.
The cells were further cultured for 20 days to obtain stable transformants.
A single clone was also obtained from the transformed cells.
Subculture in RPMI 1640 medium containing 1000 μg/ml G418 and 10% fetal bovine serum.
(3) Expression levels of poly-N-acetyllactosamine glycans in each transformed cell line
Measurement The following is an anti-i antibody (OSK28) that recognizes poly-N-acetyllactosamine glycans.
) is shown below. HCT-G6 or pCXN2-G6 prepared in (2) above was introduced.
15 cells (5 x 10 each⁶) in 3 ml of phosphate buffer PBS (8 g/l Na
Cl, 0.2g/l KCl, 1.15g/l Na₂HPO₄(Anhydrous), 0.
2g/l KH₂P.O.₄) and washed with the above cells (approximately 1 x 10⁶pcs) into a microtube (1.5 ml: Eppendorf
The cells were then transferred to a microcentrifuge (manufactured by Orf) and centrifuged (550 x g, 7 minutes) to collect the cells. The cells were then soaked in 0.9 ml of PBS containing 0.1% sodium azide (A-PBS
:8g/l NaCl, 0.2g/l KCl, 1.15g/l Na₂HPO
₄(anhydrous), 0.2g/l KH₂P.O.₄, 0.1% sodium azide)
Afterwards, the washed cells were treated with poly-N-acetate diluted to 10 μg/ml in A-PBS.
100 μl of antibody (OSK28) that recognizes tillactosamine sugar chains (purified antibody described below)
The antibody (20-fold diluted) was added to the suspension and allowed to react for 30 minutes in the dark at 4°C. OSK28 (purified antibody) was kindly provided by Dr. Junko Takahashi of Research Division 1, Osaka Red Cross Blood Center.
OSK28 is a human lysosomal protein that produces anti-Tja and anti-i antibodies.
Immortalized B cell lines established from lymphocytes using the EBV-hybridoma method
The purified OSK used in this experiment is a human monoclonal antibody (IgM antibody).
28 is a method for culturing the B cell line in large quantities and then culturing the B cell line in the manner described in (i) of (6) of the embodiment of the present invention.
The antibody was purified using the method described above. After the reaction, the cells were washed twice with 3 ml of A-PBS and diluted to 10 μg/ml.
FITC-labeled anti-human IgM antibody (manufactured by Medical and Biological Laboratories (MBL)) was added to 100
After the reaction, the cells were suspended in 3 ml of A-PB
The cells were washed twice with PBS and suspended in 200 μl of 0.5% paraformaldehyde in PBS.
The cells were then analyzed by FACS (Epix Elite Flow Cytometer).
In a control experiment, A-PBS was used instead of the antibody.
Similar analysis was performed using the G6 polypeptide expression plasmid (pCXN2-G6) or a control
HCT-15 cells transfected with the plasmid pCXN2 were treated with various anti-glycoprotein antibodies (O
SK28, KM93, PM81, CA19-9, KM231, TT42, or
7LE) and analyzed using FACS.
The results are shown in Figure 1. FACS analysis using indirect fluorescent antibody staining was performed according to a conventional method [J. Biol.
Chem.,274, 12499-12507 (1999)]. In cells transfected with pCXN2-G6, the expression level was higher than in cells transfected with pCXN2.
Compared with the control group, the reactivity to OSK28 was increased (Fig. 1).
By expressing the 6 polypeptide in HCT-15 cells, the glycoprotein on the cell surface was
Poly-N-acetyllactosamine sugar chains are newly formed on the sugar chains of proteins or glycolipids.
This means that it was synthesized. On the other hand, CA19-9 or KM23, which are antibodies against sialyl Lewis a sugar chains,
When fluorescent staining was performed using 1, HCT-15 containing pCXN2-G6
The reactivity to the antibody was not changed between HCT-15 cells and HCT-15 cells transfected with pCXN2.
HCT-15 cells express α1,3/1,4-fucosyltransferase.
(Fuc-TIII) and the cells express β1,3-galactosyltransferase.
When the enzyme is expressed, the sialyl Lewis a sugar chain detected by the above antibody is synthesized.
is known [J. Biol. Chem.,274, 12499-12507
(1999)]. Therefore, these results indicate that the G6 polypeptide is GlcNAcβ1,
This indicates that the antibody does not have 3-galactosyltransferase activity. Similarly, KM93, an antibody against sialyl Lewis x glycans,
PM81, an antibody against Lewis b sugar chains; TT42, an antibody against Lewis b sugar chains; or
When fluorescent staining was performed using 7LE, an antibody against Lewis a glycans, pC
HCT-15 cells transfected with XN2-G6 and HCT-15 cells transfected with pCXN2
The antibody reactivity in the cells did not change (Figure 1). (4) Experiments using glycan synthesis inhibitors G6 polypeptide is involved in the synthesis of glycoprotein glycans or glycolipid glycans.
To investigate whether the inhibitors of glycan synthesis were effective, an experiment was carried out using the inhibitors of glycan synthesis.
The obtained pCXN2-G6-transfected HCT-15 cells (single clones) were
After culturing for 5 days in the presence of various glycosylation inhibitors, the cells were analyzed by FACS using OSK28.
As an inhibitor of O-linked glycans of glycoproteins, benzyl-α-
GalNAc (SIGMA) was used at a concentration of 4 mmol/L.
As an inhibitor of N-linked glycans, swainsonine, a mannosidase II inhibitor,
(Swainsonine: manufactured by Seikagaku Corporation) at a concentration of 10 μg/ml
As an inhibitor of glycolipid sugar chains, a glucosylceramide synthase inhibitor was used.
D-PDMP (D-threo-1-phenyl-2-decanoyl
lamino-3-morpholino-1-propanol: Matre
(manufactured by YA Co., Ltd.) was used at a concentration of 10 μmol/L.
As an active control, L-PDMP (L-threo-1-phenyl-
2-decanoylamino-3-morpholino-1-propa
nol: manufactured by Matreya) was used at a concentration of 10 μmol/L.
The results are shown in Figure 2. As a control, the vector (
Indirect fluorescence using OSK28 on HCT-15 cells transfected with pCXN2
The results of antibody staining are also shown. When cultured in the presence of the sugar chain synthesis inhibitor Benzyl-α-GalNAc,
The reactivity to the OSK28 antibody was significantly reduced compared to when it was not present.
The poly-N-acetylglucosamine observed in HCT-15 cells expressing G6 polypeptide
of tillactosamine sugar chainsde novoThe synthesis is mainly on the O-linked glycans of glycoproteins.
On the other hand, even in swainsonine-treated cells,
Since the reactivity to SK28 antibody was reduced,
The poly-N-acetyllactosamine sugar chains observed in HCT-15 cellsde
novoThis suggests that synthesis also occurs on the N-linked glycans of glycoproteins.
From the above results, it is concluded that when highly expressed in animal cells, the G6 polypeptide acts as a glycoprotein.
It is thought that poly-N-acetyllactosamine sugar chains can be synthesized on protein sugar chains.
Also, glycoproteins and oligosaccharides secreted from cells expressing G6 polypeptide are
It is thought that poly-N-acetyllactosamine sugar chains are newly synthesized in the sugar chains of
Therefore, cells expressing the G6 polypeptide can be used as a host to produce useful glycoproteins.
By secreting and producing the glycoprotein, the glycoprotein to be secreted is poly-N-acetyl
It is possible to add a sugar chain containing lactosamine. Example 7: In human cultured cells transfected with an expression plasmid for G6 polypeptide
Measurement of β1,3-N-acetylglucosaminyltransferase activity The expression plasmid for the G6 polypeptide obtained in Example 6(1) was introduced.
Using cell extracts from stable transformants (Namalwa cells), β1,3-N
The acetylglucosaminyltransferase activity was examined. (1) Activity measurement using 2-aminobenzoated oligosaccharides as a substrate The transformed cells (approximately 2 x 10⁷pcs) into microtubes
The mixture was taken into a tube (1.5 ml, manufactured by Eppendorf) and centrifuged (550 × g,
The cells were collected by centrifugation for 7 minutes. The cells were washed with 0.9 ml of PBS, and then the washed cells were
The purified cells were cultured in 20 mmol/L HEPES (pH 7.2), 2% Triron X-
The mixture was suspended in a solution (100 μl) of 100 and then ultrasonicated (BioRupto
The cells were disrupted using a centrifuge tube (Cosmo Bio). After standing at 4°C for 1 hour,
The supernatant was obtained by centrifugation (550×g, 7 minutes).
Using the above enzyme sample and a 2-aminobenzene-modified sugar chain substrate, β1,3-N-
Acetylglucosaminyltransferase activity was measured. 2-Aminobenzene-modified glycan substrates were prepared using Sigma-Aldrich 2AB Glycan.
Labeling kit (Oxford Glycoscience)
) according to the kit instructions.
Lacto-N-neotetraose, Galβ1-4GlcNAcβ1-
3Galβ1-4Glc; hereinafter abbreviated as LNnT) and Galβ1-4Glc
NAcβ1-3Galβ1-4GlcNAc (hereinafter sometimes abbreviated as 2LN)
The 2-aminobenzene derivative of LNnT was used as the substrate.
2LN was purchased from Seikagaku Corporation.
The activity was measured by a known method [FEBS,462, 289 (1999), J. Bio
l. Chem.,269, 14730-14737 (1994), J. Biol
Chem.,267, 23507 (1992), J. Biol. Chem. ，
267, 2994 (1992)] was used.
Liquid [150 mmol/l MOPS (pH 7.5), 50 mmol/l UDP
-GlcNAc (manufactured by SIGMA), 20 mmol/L sodium cacodylate (
pH 7.2), 0.4% Triron CF-54, 10 mmol/l MnC
l₂, 15 mmol/l 2-aminobenzened sugar chain substrate, the above cell extract (protein
After incubation at 37°C for 16 hours, the product was analyzed by high performance liquid chromatography.
The protein concentration of the cell extract was detected by HPLC.
Protein Assay Kit (BIO RAD) was used, and the kit was
The assay was performed according to the manufacturer's instructions. Assay solutions containing UDP-GlcNAc (sugar donor) and those without were used.
After reaction with the solution, analysis was performed by HPLC to identify the enzyme containing UDP-GlcNAc.
The peak that appeared only in the assay solution was determined to be the product. After the reaction, the assay solution was heated at 100°C for 5 minutes, and then diluted with 50% pure water for HPLC.
The supernatant was collected by centrifugation at 10,000×g for 5 minutes.
Add 50 μl of pure water for HPLC to the tube containing the solution to wash the tube.
After washing, the mixture was centrifuged at 10,000 x g for 5 minutes to obtain the supernatant, which was then combined with the first supernatant.
The supernatant was then loaded onto an Ultrafree-MC column (Millipore
After passing the mixture through an Ultrafried Column (manufactured by Ultrafried Column), a portion (10 μl) was subjected to HPLC.
The ee-MC column was used according to the attached instructions. HPLC was performed using a TSK-gel ODS-80Ts column (4.6
× 300 mm; Tosoh), and 0.02 mol/l containing 7% methanol as eluent
Ammonium acetate buffer (pH 4.0) was used, and the elution temperature was 50°C and the flow rate was 1 ml/
The reaction was carried out under conditions of 2 min. Product detection was performed using a fluorescence spectrophotometer FP-920 (manufactured by JASCO Corporation).
) (excitation wavelength 330 nm, emission wavelength 420 nm). Products were identified based on whether their elution time matched that of standard glycans.
As a standard sugar chain, 2-aminobenzene-modified GlcNAcβ1-3G
alβ1-4GlcNAcβ1-3Galβ1-4Glc was used. Product quantification was performed using 2-aminobenzene-modified glucose polymers (Oxfor
d Glycoscience) was used as a standard, and the fluorescence intensity was compared.
This was done by comparing the control plasmid (pAMo) or the G6 polypeptide expression plasmid.
The cells of stable transformed cells (Namalwa cells) into which the vector (pAMo-G6) was introduced were
The activity was measured using the cytosolic extract.
β1-4GlcNAcβ1-3Galβ1-4Glc)
nT) was almost 0% in cells transfected with the control plasmid.
In contrast, in cells transfected with an expression plasmid for G6 polypeptide, the percentage was 6.11%.
In addition, the product (GlcNAcβ1-3Galβ1-4 ...
The substrate (Galβ1-4Acβ1-3Galβ1-4GlcNAc) was converted to
The ratio of GlcNAcβ1-3Galβ1-4GlcNAc was 1:1.
The expression of G6 polypeptide was almost 0% in the cells transfected with the plasmid.
In cells transfected with the present plasmid, the percentage increased to 3.95%.
In cells transfected with the peptide expression plasmid, the control plasmid was transfected.
Increased β1,3-N-acetylglucosaminyltransferase activity compared to untreated cells
It was found that the G6 polypeptide is a novel β1,3-N-acetylglucosamine.
This result was demonstrated using the G6 polypeptide.
The galactose residue at the non-reducing end of the sugar chain is converted to N-acetylglucosamine.
This indicates that the enzyme is capable of synthesizing glycans in which α-glucan is attached via a β1,3 bond. (2) Activity measurement using glycolipids as substrates According to a known method [FEBS, 462, 289 (1999), J. Biol. Chem.
m.,269, 14730-14737 (1994), J. Biol. Chem
．267, 23507 (1992), J. Biol. Chem. ，267, 2
994 (1992)], the β1, β2, and β3 of G6 polypeptide were synthesized using glycolipid as a substrate.
3-N-acetylglucosaminyltransferase activity was measured.
Reaction solution [150 mmol/L sodium cacodylate (pH 7.2), 10 mm
ol/l UDP-GlcNAc (manufactured by SIGMA), 480 μmol/l U
DP-[¹⁴C]GlcNAc (Amersham), 0.4% Triro
n CF-54, 10 mmol/l MnCl₂, 250 μmol/l glycolipid,
The cells were incubated at 37°C for 16 hours in the cell extract (20 μg of protein) prepared in (1) above.
As glycolipids, lactosylceramide
ide: manufactured by SIGMA) and paragloboside
(Provided by Professor Yasunori Kushi, Tokyo Medical and Dental University) was used.
200 μl of 1/1 KCl was added, and the mixture was centrifuged briefly to obtain the supernatant.
After washing once with ethanol, equilibrate with 10 ml of 0.1 mol/l KCl twice.
Sep-Pak plus C18 Cartridge (Waters
The supernatant was passed through a cartridge containing 10 ml of H
After washing the cartridge twice with pure water for PLC, the adsorbed sugars were removed with 5 ml of methanol.
The lipids were eluted. The eluate was concentrated to about 10 μl using a vacuum dryer, and the concentrated solution was
TLC plate (HPTLC plate Silica gel 60:ME
RCK) and mixed with chloroform:methanol:water (0.2% CaCl
₂The TLC was developed using a developing solvent with a composition of 65:35:8.
The plate was spread to 5 mm from the top edge, and after drying, the plate was
The enzyme was incorporated into glycolipids using the analyzer BAS2000 (Fuji Photo Film Co., Ltd.).
The amount of radiation absorbed was measured. Control plasmid (pAMo) or G6 polypeptide expression plasmid
The cells of stable transformed cells (Namalwa cells) into which the vector (pAMo-G6) was introduced were
When lactosylceramide was used as a substrate, the activity was measured using the cellular extract.
, whereas no activity was detected in cells transfected with the control plasmid.
In cells transfected with an expression plasmid for the G6 polypeptide, clear activity was detected.
(Product conversion efficiency: 0.125%). When paragloboside was used as a substrate,
Even in cells transfected with the control plasmid, the activity was weak (0.01%).
The cells transfected with the G6 polypeptide expression plasmid showed a significant increase in the number of G6 polypeptides (0.006%).
In the cells, the activity was clearly increased (product conversion efficiency: 0.126%).
From the above results and the results of (1) above, it is concluded that the G6 polypeptide is capable of synthesizing lactosylceramide and
A novel β1,3-N-acetylglucosamine transferase using paragloboside as a substrate
It was proved that the enzyme is a transferase.
The activity of the 6 polypeptides was taken as 100%.
The activity was 96% when G6 polypeptide was used. The above results also support the idea that lactosylceramide and paraglucan were synthesized using G6 polypeptide.
Galactose residues present at the non-reducing ends of sugar chains such as loboside are N-acetylated.
This demonstrates that it is possible to synthesize glycolipids to which glucosamine is attached via a β1,3 bond. Example 8: Analysis of FLAG peptide-fused G6 polypeptide using insect cells as a host
Secretory production The cloned β1,3-N-acetylglucosaminyltransferase G6 is one of
The following sequence shows a cytoplasmic region consisting of 14 amino acids at the N-terminus, followed by 18 amino acids:
a hydrophobic membrane-binding region consisting of amino acids; a stem region consisting of at least 12 amino acids;
It was thought to consist of the cytoplasmic domain consisting of 14 amino acids at the N-terminus of the G6 polypeptide, and most of the remaining C-terminal region including the catalytic domain.
The membrane-binding region consists of 3 or 6 amino acids, and a part of the stem region
and replacing the immunoglobulin signal sequence and FLAG in the removed region.
We attempted to secrete and express G6 polypeptide by adding a peptide. (1) Construction of FLAG peptide-fused secretion vector pAMoF2 for animal cells FLAG peptide having the amino acid sequence represented by SEQ ID NO: 13 was added to any desired transcription factor.
The secretion vector pAMoF2 was used for secretory expression of the protein in the form of a protein attached to the N-terminus.
pAMo was created.HindIII andAspBy cutting at 718, it becomes about 8.7k
b'sHindIII-Asp718 fragments were obtained.HindIII cleavage site and
AspThe following six types of DNA [I] were used as linkers for linking the 718 cleavage sites:
IgK-1 (nucleotide sequence: SEQ ID NO: 14), IgK-2 (nucleotide sequence: SEQ ID NO: 15)
, IgK-3 (nucleotide sequence: SEQ ID NO: 16), IgK-4 (nucleotide sequence: SEQ ID NO: 1
7), IgK-5 (nucleotide sequence: SEQ ID NO: 18), IgK-6 (nucleotide sequence: SEQ ID NO:
No. 19)] was synthesized. The linker constructed by these DNAs was
Encoding the immunoglobulin κ signal sequence and FLAG peptide,PmaCI
,StuI,SnaEach restriction enzyme cleavage site of BI is incorporated.
A was prepared using the 380A DNA synthesizer manufactured by Applied Biosystems.
The synthesized DNA was purified using T4 polynucleotide kinase (Takara Shuzo Co., Ltd.).
(hereinafter the same) and then used after phosphorylation. The six types of phosphorylated synthetic DNA obtained above and the approximately 8.7 kbHindIII-
AspPlasmid pAMoF2 was constructed by ligating the 718 fragment. (2) Construction of plasmid pAMoF2-i52S A DNA having the base sequence represented by SEQ ID NO: 20 was used as a PCR primer.
A (hereinafter referred to as C12-7) and having the base sequence represented by SEQ ID NO: 21
DNA (hereafter referred to as C12-9) was synthesized (from Sawasdee Technology
(It is also possible to purchase it.) C12-7 hasBamThe HI site is C12-9No
tIt is designed to introduce an I site. PCR was performed using a kit manufactured by Takara Shuzo (GeneAmp^TM DNA Amplif
cation Reagent Kit with AmpliTaq^TM
Recombinant Taq DNA Polymerase
The reaction mixture was prepared according to the kit instructions, and the reaction mixture was run in a DNA thermal cycler (
PERKIN ELMER CETUS DNA Thermal Cycle
r; sold by Takara Shuzo Co., Ltd.) was used, and the temperature was set at 94°C for 30 seconds, 65°C for 1 minute, and 72°C for 2 minutes.
After 10 cycles of reaction, the reaction was further carried out at 72°C for 7 minutes.
10 ng of the plasmid pAMo-i (JP 11-236398 A) was used.
A DNA fragment of approximately 1.1 kb was obtained by PCR. The PCR-amplified DNA fragment of approximately 1.1 kb was cloned into the T-vector pT7Blue (Nov
By ligating this with the plasmid pT7B-i52S No.
3 was constructed. Next, the plasmid pAMoF2-i52S was constructed. pAMoF2StuI andBanIII to give a fragment of about 7.2 kb.StuI-
BanFragment III was obtained.BanIII andNotCut at I and about
1.7 kbBanIII-NotThe I fragment was obtained. pT7B-i52S N
No. 3BamAfter digestion with HI, E. coli DNA polymerase I Klenow fragment was used.
ItBamThe 5' protruding ends generated by HI digestion were converted to blunt ends, and thenN
otBy digesting with .I, a fragment of approximately 1.1 kb was obtained.BamHI (blunt end)-Not I fragment was obtained. The approximately 7.2 kb fragment obtained aboveStuI―BanFragment III, approximately 1.7kb
ofBanIII-NotI fragment and approximately 1.1 kbBamHI (blunt end)-
NotThe I fragment was ligated to construct the plasmid pAMoF2-i52S. (3) Construction of Plasmids pBS-G6sec1 and pBS-G6sec2 The region of the G6 polypeptide believed to have catalytic activity (position 36 of SEQ ID NO: 1)
from the aspartic acid at position 378 to the isoleucine at position 378 of SEQ ID NO: 1
DNA encoding the 9th isoleucine to the 378th isoleucine
The fragments were subcloned. CB-543 having the base sequence represented by SEQ ID NO: 22 and CB-544 having the base sequence represented by SEQ ID NO: 23 were
CB-545 having the base sequence shown in Example 3 was used as a primer.
PCR was performed using pBS-G6 as a template to obtain the 36th base of SEQ ID NO: 1.
Approximately 1.0 kb of the sequence encoding from aspartic acid to isoleucine at position 378
DNA fragments were prepared.BamThe HI cleavage site is CB-54
In 5XbaThe PCR amplified fragment wasBamHI andXb
aAfter digestion with , the vector pBluescript SK(-)BamHI-X
bapBS-G6sec1 was constructed by inserting CB-544, which has the base sequence represented by SEQ ID NO: 24, and CB-544, which has the base sequence represented by SEQ ID NO: 25.
CB-545 having the base sequence shown in Example 3 was used as a primer.
PCR was performed using pBS-G6 as a template to obtain the 39th isoform of SEQ ID NO: 1.
Approximately 1.0 kb of DNA encoding leucine to isoleucine at position 378
The fragments were prepared.BamThe HI cleavage site is
XbaThe PCR amplified fragment wasBamHI andXbaI
After digestion, the vector pBluescript SK(-)BamHI-XbaI
pBS-G6sec2 was constructed by integrating this between the two. PCR was performed using Platinum Pfx DNA polymerase (GIB
The template used was 1 ng of the plasmid pBS-G6.
50 μl of the PCR reaction solution was heated at 94°C for 2 minutes, and then heated at 94°C for 20 minutes.
One cycle consisted of a reaction at 55°C for 45 seconds, 55°C for 45 seconds, and 68°C for 2 minutes.
Five cycles of reaction were performed using Platinum Pfx DNA pol.
The instructions for the .ymerase kit were followed. DNA fragments incorporated into pBS-G6sec1 and pBS-G6sec2
The base sequence of each fragment was determined to confirm the absence of PCR errors. (4) To secrete and express the FLAG peptide-fused G6 polypeptide in insect cells,
Creation of recombinant viruses The DNA encoding the target protein is inserted into a special vector called a transfer vector.
The first step is to integrate the target DNA into a suitable plasmid (step 1), and the second step is to integrate the target DNA prepared in step 1 into a suitable plasmid (step 2).
The transferred vector and wild-type virus were cotransfected into insect cells.
and obtaining a recombinant virus by homologous recombination (step 2).
The recombinant virus was produced by the following process.
Gold Starter Kit (product number PM-21001K) was used.
The procedure was carried out according to the manual: (Step 1) DNA encoding the FLAG peptide-fused secreted G6 polypeptide was prepared.
Integration into a transfer vector Transfer vector pVL1393 (Pharmingen)Ba
mHI site andNotBetween the I site, a FLAG peptide-fused secreted G6 polypeptide
Plasmid pVL1393-F2G6s incorporating DNA encoding the peptide
ec1 and pVL1393-F2G6sec2 were constructed. pAMoF2-i52S prepared in (2) above was purified with restriction enzymesHindIII andN
otI and a 1.2 kb fragmentHindIII-NotI fragment was obtained. pVL1393 wasBamHI andBstPI, and 3.2 kb
ofBamHI-BstThe PI fragment was obtained. pVL1393 wasNotI andBstPI and a 6.4 kb fragment
NotI-BstThe PI fragment was obtained. BamHI site andHinIt is arranged as a linker for connecting the dIII sites.
The DNAs shown in columns 26 and 27 were synthesized and used to prepare T4 polynucleotides.
The 5' end was phosphorylated using kinase. By linking the above three fragments with a linker, pVL1393-F2i52S was obtained.
2 was constructed. pVL1393-F2i52S2BamHI andNotCut at I and about 9.6
kbBamHI-NotThe pB I fragment was obtained.
S-G6sec1 is digested with a restriction enzymeBamHI andNotI and approximately 1.0 kbB
amHI-NotBy combining the above two fragments, the plus
The plasmid pVL1393-F2G6sec1 was constructed. pVL1393-F2i52S2BamHI andNotCut at I and about 9.6
kbBamHI-NotThe pB I fragment was obtained.
S-G6sec2 is digested with a restriction enzymeBamHI andNotI and approximately 1.0 kbB
amHI-NotBy combining the above two fragments, the plus
The vector pVL1393-F2G6sec2 was constructed. (Step 2) Preparation of recombinant virus Cultured in TNM-FH insect medium (Pharmingen).
The filamentous baculovirus was transfected into the cultured insect cells Sf9 (Pharmingen).
DNA [BaculoGold Baculovirus DNA (BaculoGold
baculovirus DNA), manufactured by Pharmingen] and the above
The prepared plasmid (pVL1393-F2G6sec1 or pVL1393
-F2G6sec2) was transfected using the lipofectin method [protein nucleic acid enzyme,37, 2701 (
Recombinant baculovirus was generated by introducing the vector into the host cell using the recombinant vector.
The following recombinant baculoviruses derived from pVL1393-F2G6sec1 were prepared:
The method for producing the recombinant baculovirus derived from pVL1393-F2G6sec2 is described below.
The preparation of the vector was carried out in the same manner. 1-5 μg of pVL1393-F2G6sec1 and 15 ng of filamentous baculoplasmic
The viral DNA was dissolved in 12 μl of distilled water and then infected with Lipofectin (GIBCO BR).
A mixture of 6 μl (6 μg) of the 100 μL PBS solution (manufactured by L Co.) and 6 μl of distilled water was added, and the mixture was left to stand at room temperature for 1
Let it sit for 5 minutes. Approx. 2 x 10⁶100 Sf9 cells were cultured in 2 ml of Sf900-II medium (GIBCO
The cells were suspended in a 35 mm diameter plastic cell culture dish.
After that, pVL1393-F2G6sec1, linear baculovirus DNA, and
The entire volume of the mixed solution of ribosomal protein and lipofectin was added, and the mixture was cultured at 27°C for 3 days. 1 ml of culture supernatant containing the recombinant virus was collected from the culture medium. The Petri dish from which the culture supernatant was collected was filled with fresh TNM-FH insect medium.
After the incubation, 1 ml of the culture medium was added and the mixture was further cultured at 27°C for 4 days.
An additional 1.5 ml of culture supernatant containing the virus was obtained. (5) Obtaining recombinant virus solution Approximately 8 x 10⁶100 Sf9 cells were cultured in 5 ml of EX-CELL 400 medium (JRH Co., Ltd.).
Suspended in 25 cm²The flask (manufactured by Greiner) was placed in a flask and heated at room temperature for 30
After leaving the flask for 1 minute to allow the cells to adhere to the flask, the supernatant was removed and 1 ml of
EX-CELL 400 medium and the culture containing the recombinant virus obtained in (4) above
1 ml of supernatant was added. After addition, the mixture was gently shaken at room temperature for 1 hour to thoroughly mix the cells and virus.
After contact with the spores, 4 ml of TNM-FH insect medium was added and the mixture was incubated at 27°C for 4
The culture medium was centrifuged at 1500 x g for 10 minutes to isolate the recombinant virus.
5.5 ml of infected Sf9 cells and recombinant virus solution were obtained. Approximately 2 x 10⁷Sf9 cells were suspended in 15 ml of EX-CELL 400 medium.
, 75cm²The flask (Greiner) was placed in the flask and left at room temperature for 30 minutes.
After the cells were allowed to adhere to the flask, the supernatant was removed and 5 ml of EX-CE was added to the flask.
LL400 medium and 1 ml of the recombinant virus solution obtained above were added. After addition, the cells and virus were thoroughly mixed by gently shaking at room temperature for 1 hour.
After contact with the spores, 10 ml of TNM-FH insect medium was added and the mixture was incubated at 27°C.
The culture was cultured for 4 days. The culture medium was centrifuged at 1500×g for 10 minutes to obtain
Sf9 cells infected with the recombinant virus and 15 ml of the recombinant virus solution were obtained.
The virus titer of the recombinant virus solution can be calculated using the following method.
(Pharmingen BaculoGold Starter Kit Manual
(based on the above). Approx. 6 x 10⁶100 Sf9 cells were suspended in 4 ml of EX-CELL 400 medium,
Place in a 60 mm diameter plastic petri dish for cell culture and leave at room temperature for 30 minutes.
After the cells were attached to the dish, the supernatant was removed and EX-CELL4 was added to the dish.
400 μl of EX-CELL 400 medium and 10⁻⁴or 10⁻⁵
Add 100 μl of the diluted recombinant virus solution to the dish. After addition, gently shake the dish at room temperature for 1 hour to allow the cells and virus to mix.
After contact, remove the medium from the dish and add 2% low-melting-point agarose [
Agarplaque Agarose; Ph
2 ml of EX-CELL 400 medium (kept at 42°C) containing
A mixture of 2 ml of TNM-FH insect medium (kept at 42°C) and 2 ml of TNM-FH insect medium (kept at 42°C)
Pour the contents into the dish and leave it at room temperature for 15 minutes. After leaving it, wrap vinyl tape around the dish to prevent it from drying out, and store it in a sealable plastic container.
The dish is placed in a stick container and cultured at 27°C for 5 days. After culture, 1 ml of PBS buffer containing 0.01% neutral red is added to the dish.
Add 1 ml of FLAG peptide to the culture medium, and then culture for another day. Then, count the number of plaques that appear. (6) Secretion and purification of FLAG peptide-fused G6 polypeptide Plasmid pVL1393-F2G6sec1 or pVL1393-F2G
The G6 polypeptide encoded by the recombinant virus derived from 6sec2 contains the FLAG peptide.
Since the protein is secreted and expressed as a fusion protein with the anti-FLAG peptide,
1 affinity gel (Anti-FLAG M1 Affinity Gel
(Cosmo Bio Co., Ltd.) Recombinant viruses derived from pVL1393-F2G6sec1 consist of eight amino acids.
The C-terminus of the FLAG peptide is connected to the G6 polynucleotide via Gly and Ser residues.
The putative catalytic region of the peptide (from aspartic acid at position 36 to aspartic acid at position 378 of SEQ ID NO: 1)
The recombinant virus derived from pVL1393-F2G6sec2 can secrete and produce polypeptides fused with 8 amino acids to the 1st isoleucine.
The C-terminus of the FLAG peptide is connected to the G6 polynucleotide via Gly and Ser residues.
The putative catalytic region of the peptide (from the 39th isoleucine to the 378th isoleucine of SEQ ID NO: 1)
Polypeptides fused with up to 10 isoleucine can be secreted and produced. Approximately 2 x 10⁷100 Sf21 cells were suspended in 15 ml of EX-CELL 400 medium.
75cm²Place in a flask (Greiner) and leave at room temperature for 30 minutes.
After the cells were allowed to adhere to the flask, the supernatant was removed and the flask was filled with 4 ml of EX-CE.
LL400 medium and 1 ml of the recombinant virus solution obtained in (5) above were added.
After addition, gently shake at room temperature for 1 hour to thoroughly mix the cells and virus.
After contact with the spores, 10 ml of TNM-FH insect medium was added and the mixture was incubated at 27°C.
The culture was cultured for 4 days. The culture medium was centrifuged at 1500×g for 10 minutes to obtain
15 ml of culture supernatant was obtained. 15 ml of the culture supernatant obtained above was added with sodium azide, sodium chloride, and
and calcium chloride at final concentrations of 0.1%, 150 mmol/L, and
After adding it to 2 mmol/l, anti-FLAG M1 affinity gel
(Anti-FLAG M1 Affinity Gel; manufactured by Cosmo Bio Co., Ltd.)
) was added to the tube and the mixture was stirred slowly overnight at 4°C. After stirring, the mixture was centrifuged at 160 x g for 10 minutes to separate the anti-FLAG M1 antibody.
The affinity gel was collected and washed with 50 mmol/L Tris-HCl (pH 7
4), 150 mmol/l sodium chloride, 1 mmol/l calcium chloride
The gel was washed twice with 1 ml of a buffer solution containing 50 mmol/L Tris-HCl (pH 7.4), 150 ml of
30 μl of buffer solution containing 2 mmol/l sodium chloride and 2 mmol/l EDTA
The proteins adsorbed on the gel were dissolved by adding HCl and treating at 4°C for 30 minutes.
Then, the mixture was centrifuged at 160×g for 10 minutes to obtain the supernatant.
. The gel was again diluted with 50 mmol/L Tris-HCl (pH 7.4), 150 mmol
30 μl of a buffer solution containing 2 mmol/l sodium chloride and 2 mmol/l EDTA was added.
After heating at 4°C for 10 minutes, the mixture was centrifuged at 160 x g for 10 minutes.
The supernatant was then obtained. The above procedure was then repeated three times, and the total elution was
85 μl of eluate was obtained. The eluate was diluted with 1 mol/L calcium chloride to a final concentration of 4 mmol/L.
Sodium was added. After performing SDS-PAGE using 8 μl of the eluate prepared in this way,
Silver staining or Western blotting using anti-FLAG antibody was performed.
Staining was performed using Silver Staining II Kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.).
The method was performed according to the kit's instructions. Western blotting using anti-FLAG antibody
The tag is Yeast Amino-Terminal Flag Express
The procedure was carried out using the ION KIT (manufactured by SIGMA) according to the kit's instructions.
The results of silver staining or Western blotting using anti-FLAG antibody were compared in the third
Figure 3A shows the expression of the FLAG peptide-fused secreted G6 polypeptide.
Plasmids [pVL1393-F2G6sec1 (lanes 3 and 4) or pVL
1393-F2G6sec2 (lanes 5 and 6)
The culture supernatant of Sf21 cells (lanes 4 and 6) was purified with anti-FLAG M1 affinity chromatography.
Using a gel, the FLAG peptide-fused secreted G6 polypeptide (G6sec1) was analyzed.
or G6sec2) were purified (lanes 3 and 5) and analyzed by SDS polyacrylamide gel electrophoresis.
This figure shows the results of silver staining after subjecting the control to gel electrophoresis.
Sf21 cells infected with recombinant virus derived from plasmid pVL1393
A sample was prepared in the same manner (lane 1) from the culture supernatant of (lane 2).
indicates the location and size of the secreted G6 polypeptide produced. Figure 3B shows the expression plasmid for the FLAG peptide-fused secreted G6 [pVL1
393-F2G6sec1 (lane 2) or pVL1393-F2G6sec
2 (lane 3)] from the culture supernatant of Sf21 cells infected with the recombinant virus
Anti-FLAG M1 affinity gel was used to detect FLAG peptide-fused secreted G6
The polypeptide (G6sec1 or G6sec2) was purified and purified by SDS polyacrylamide gel electrophoresis.
After subjecting the sample to amide gel electrophoresis, Western blot analysis was performed using an anti-FLAG peptide antibody.
This figure shows the results of blotting.
The same was obtained from the culture supernatant of Sf21 cells infected with the recombinant virus derived from pVL1393.
The arrow indicates the secreted G6 polynucleotide produced (lane 1).
The position and size of the peptide are shown. As a result, when the recombinant virus derived from pVL1393-F2G6sec1 was infected,
When an eluate prepared from the culture supernatant of Sf21 was used, the
A broad band of D was observed. The size of the main band was approximately 43 kD.
The recombinant virus contained the following at the C-terminus of the 8-amino acid FLAG peptide:
The putative catalytic region of the G6 polypeptide (SEQ ID NO: 1) is bound to the Gly and Ser residues.
1 from the 36th aspartic acid to the 378th isoleucine]
Since the polypeptide is thought to be secreted, the polypeptide calculated from the amino acid sequence
The molecular weight of the detected band was calculated to be 40.95 kD.
The larger value compared to the previous value is thought to be due to the addition of a sugar chain.
It has four putative N-linked glycan attachment sites. The broad band is due to the presence of the attachment sites.
This indicates that there are polypeptides with different numbers and sizes of sugar chains. Sf2 infected with recombinant virus derived from pVL1393-F2G6sec2
When the eluate prepared from the culture supernatant of No. 1 was used, a blown protein of about 41 to 47 kDa was obtained.
A major band was observed. The size of the main band was approximately 43 kD.
The recombinant virus contains a Gly residue at the C-terminus of the 8-amino acid FLAG peptide.
and Ser residues, the putative catalytic region of the G6 polypeptide [position 39 of SEQ ID NO: 1]
a polypeptide fused with the 378th isoleucine to the 378th isoleucine
Therefore, the polypeptide calculated from the amino acid sequence
The molecular weight of the detected band is 40.6 kD.
The reason for this is thought to be the addition of sugar chains.
It has four putative attachment sites. The band is broad due to the number of added glycans and
This indicates the presence of polypeptides of different sizes. On the other hand, Sf infected with a recombinant virus derived from the vector pVL1393
When the eluate prepared from the culture supernatant of 21 was used, the band was not detected.
Band intensity detected by Western blotting using anti-FLAG antibody
was detected in the eluate from the recombinant virus derived from pVL1393-F2G6sec1.
When the intensity of the band emitted was set to 1, the intensity of the band derived from pVL1393-F2G6sec2 was
The intensity of the band detected in the eluate derived from the recombinant virus was 1.05. Based on these results, it is possible to express the FLAG peptide-fused G6 polypeptide using insect cells.
and the polypeptide can be secreted and produced by anti-FLAG M1 affinity chromatography.
It was shown that the purified product can be easily purified using a gel. Example 9: Using a FLAG peptide-fused G6 polypeptide produced in insect cells
Activity study The FLAG peptide-fused G6 polypeptide produced and purified in Example 8 was investigated using various substrates.
The β1,3-N-acetylglucosaminyltransferase activity of the peptide was measured.
(1) Activity measurement using 2-aminobenzylated oligosaccharides as a substrate The eluate prepared in (6) of Example 8 was used to measure the activity of FL secreted and produced in insect cells.
β1,3-N-acetylglucosamine transfer of AG peptide-fused G6 polypeptide
The enzyme activity was measured using the method described in Example 7. The substrate was oligosaccharide [LNnT, Galβ1-4GlcNAcβ1-3G
alβ1-4GlcNAc (hereinafter abbreviated as 2LN), Galβ1-4Glc
NAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNA
c (hereinafter abbreviated as 3LN), Galβ1-4GlcNAcβ1-3Galβ
1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-
4GlcNAc (hereinafter abbreviated as 4LN), Galβ1-4GlcNAcβ1
-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3
Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc (hereinafter, 5L
N), or Galβ1-4 (SO₃-6) GlcNAcβ1-3G
alβ1-4(SO₃-6) GlcNAc (hereinafter abbreviated as L2L2)
The 2-aminobenzene-conjugated oligosaccharides were used.
SIGMA 2AB glycan labeling kit (Oxford)
d Glycoscience) according to the kit instructions.
LNnT was purchased from Oxford Glycosystems.
The oligosaccharides were obtained from Seikagaku Corporation. Specifically, 20 μl of assay solution [150 mmol/l sodium cacodylate
(pH 7.2), 50 mmol/l UDP-GlcNAc (Sigma-Aldrich)
), 0.4% Triron CF-54, 10 mmol/l MnCl₂, 5
μmol/L 2-aminobenzoated sugar chain substrate, the above purified enzyme solution] at 37°C.
After 16 hours of reaction, the product was purified by high performance liquid chromatography (HPLC, details will be given later).
The enzyme was detected using the enzyme pVL1 described in Example 8 (6).
Sf21 cultures infected with recombinant viruses derived from 393-F2G6sec1
Eluate (1.5 μl) prepared from the supernatant, or pVL1393-F2G6sec
The eluate (
1.4 μl) was used. After the reaction, the assay solution was treated at 100°C for 5 minutes, and then diluted with 50% pure water for HPLC.
The mixture was centrifuged at 10,000×g for 5 minutes to obtain the supernatant.
Add 50 μl of pure water for HPLC to the tube containing the above and wash the tube.
The mixture was centrifuged at 10,000×g for 5 minutes to obtain the supernatant, which was then combined with the first supernatant.
The supernatant was then applied to an Ultrafree-MC column (Millipore).
After passing through the column, a portion (10 μl) was subjected to HPLC.
The C column was used according to the attached instructions. HPLC was performed using a TSK-gel ODS-80Ts column (4.6
× 300 mm; Tosoh), and 0.02 mol/l containing 7% methanol as eluent
Ammonium acetate buffer (pH 4.0) was used, and the elution temperature was 50°C and the flow rate was 1 ml/
The reaction was carried out under conditions of 2 min. Product detection was performed using a fluorescence spectrophotometer FP-920 (manufactured by JASCO Corporation).
) (excitation wavelength 330 nm, emission wavelength 420 nm). Produced and purified using recombinant virus derived from pVL1393-F2G6sec1
The secreted enzyme (referred to as G6sec1) and pVL1393-F2G6sec
The secreted enzyme (called G6sec2) was produced and purified using a recombinant virus derived from
As a result of measuring the activity using β1,3-N-acetylglucosamine
The conversion efficiency to the product when LNnT was used as a substrate was 1.0%.
When G6sec1 was used, the rate was 9.85%, and when G6sec2 was used, the rate was 11.2%.
The results of examining substrate specificity using G6sec1 or G6sec2 were as follows:
The results are shown in Table 1 (Table 1 Experiment 1).
The relative activity is shown with the activity of the target gene set at 100%.
The samples were prepared using recombinant virus (control virus) derived from 3.
No activity was detected. Furthermore, the purified enzyme (G6sec2) was purified again using the method shown in Example 8 (6).
The results of the substrate specificity study are shown in Table 1 (Table 1).
Experiment 2) Infection with recombinant virus derived from pVL1393-F2G6sec2
A purified enzyme (280 μl) was prepared from 30 ml of the culture supernatant of Sf21.
The assay was performed using 2-aminobenzylated LNnT as a substrate.
The relative activity is shown when the activity when LNnT was used as a substrate was taken as 100%.
The conversion efficiency to products was 26.2%. As a result, poly-N-acetyllactosamine sugar chains (2LN, 3LN, 4LN,
and 5LN) were also found to be substrates for the G6 polypeptide.
-acetyllactosamine sugar chains (2LN, 3LN) form long poly-N-acetyllactosamine chains.
Compared with the ctosamine sugar chain (4LN, 5LN), it is a substrate for G6 polypeptide.
It was also revealed that the poly-N-acetyllactose with sulfate groups attached was easily
It was also found that the tosamine sugar chain L2L2 can serve as a substrate for the G6 polypeptide. Anti-FLAG M1 affinity staining with G6sec1 or G6sec2
When the gel (gel before enzyme elution) was used as the enzyme,
Cetylglucosaminyltransferase was detected.
-The amount of gel equivalent to the eluate used in measuring acetylglucosaminyltransferase activity
The conversion efficiency when LNnT was used as a substrate was
6.64% when using G6sec1 adsorption gel, and 20.2% when using G6sec1 adsorption gel.
This result indicates that glycan synthesis is possible even when the enzyme is adsorbed onto the gel.
On the other hand, a recombinant virus derived from pVL1393 (control virus) was used.
No activity was detected in a gel prepared in the same manner. On the other hand, G6sec1 and G6sec2 reacted with GlcNAcβ1,3-galactosidase.
The β1,3-galactosyltransferase activity was not observed.
, common law [J. Biol. Chem. ，274, 12499-12507 (199
9)]. 20 μl of assay solution [14 mmol/l HEPE
S (pH 7.4), 75 μmol/l UDP-Gal (manufactured by SIGMA), 1
1 mmol/l MnCl₂, 88 pmol/L sugar chain substrate, the above purified enzyme]
The reaction was carried out at 37°C for 16 hours.
The amount was the same as that used to measure cosaminyltransferase activity.
Aminobenzene-modified GlcNAcβ1-3Galβ1-4GlcNAcβ1-3
Galβ1-4GlcNAc (hereinafter abbreviated as GlcNAc-2LN) was used.
The sugar chain was prepared by treating 2-aminobenzene-modified LN3 with β-galactosidase.
The terminal galactose residues were removed by adding the α-glucan.
100 milliunits of 2-aminobenzenated LN3 for 60 nmol
β-galactosidase (Seikagaku Kogyo Co., Ltd.) was added, and the mixture was incubated at 37°C for 16 hours.
The β-galactosidase was inactivated by heat treatment at 00°C for 5 minutes.
These results suggest that the FLAG peptide-fused G6 polynucleotide secreted and expressed in insect cells
The peptide (G6sec1 or G6sec2) is a β1,3-N-acetylglucosamine.
It has cosaminyltransferase activity but no β1,3-galactosyltransferase activity.
This result indicates that the G6 polypeptide binds to β1,3-galactose.
It was confirmed that it is not a transferase but a β1,3-N-acetylglucosaminyltransferase.
β1,3-N-acetylglucosaminyltransferase G6 was confirmed to be a FLAG
It can be secreted and produced in insect cells as a fusion protein with a peptide.
The protein can be easily purified using anti-FLAG M1 affinity gel.
The fusion protein produced was found to be poly-N-acetyllactosamine.
It was shown that it can be used to synthesize glycans such as glycans. Compared to when produced in Namalwa cells, when produced in insect cells,
It was revealed that the enzyme produced a high amount of enzyme. (2) Activity measurement using pyridylaminated oligosaccharides as substrates 30 μl of assay solution [150 mmol/L sodium cacodylate (pH 7.0)] was added.
．． 2), 50 mmol/l UDP-GlcNAc (manufactured by SIGMA), 0.4
%Triron CF-54, 10 mmol/l MnCl₂, 50 μmol/
l pyridylaminated sugar chain substrate, purified enzyme solution (G6sec2) at 37°C,
After 14.5 hours of reaction, the product was detected by HPLC.
2) was obtained according to the method described in Example 8 (6).
30 ml of culture supernatant of Sf21 infected with recombinant virus derived from 2G6sec2
The purified enzyme (280 μl) was prepared from the mixture, and 5 μl of the purified enzyme was used in the assay.
The substrates include LnNT, lacto-N-tetraose (Lacto-N-tet
raose, Galβ1-3GlcNAcβ1-3Galβ1-4Glc;
LNT), lacto-N-fucopentaose II (Lacto-N-f
ucopentaose II, Galβ1-3 (Fucα1-4) GlcNA
cβ1-3Galβ1-4Glc (hereinafter abbreviated as LNFP-II), lacto-
N-fucopentaose III (Lacto-N-fucopentaose I
II, Galβ1-4 (Fucα1-3) GlcNAcβ1-3Galβ1-4
Glc; hereinafter abbreviated as LNFP-III), lacto-N-fucopentaose V
(Lacto-N-fucopentaose V, Galβ1-3GlcNA
cβ1-3Galβ1-4(Fucα1-3)Glc; hereinafter abbreviated as LNFP-V
), and lacto-N-difucohexaose II (Lacto-N-di
fucohexaose II, Galβ1-3 (Fucα1-4) GlcNA
cβ1-3Galβ1-4(Fucα1-3)Glc; hereinafter referred to as LNDFH-II
(abbreviated as "Oxford Glycosystems") was
The substrate was fluorescently labeled with benzopyridine in accordance with a conventional method [Agri
c. Biol. Chem. ，54, 2169 (1990)]. For each substrate, an assay containing UDP-GlcNAc (sugar donor) was performed.
After reaction using the assay solution containing the α-glucan-1-phosphate buffer and the α-glucan-1-phosphate buffer, the UDP-Glc
The peak that appeared only in the assay solution containing NAc was determined to be the product. After the reaction, the assay solution was heated at 100°C for 5 minutes and then spun at 10,000 x g.
The mixture was centrifuged at 400°C for 5 minutes to obtain the supernatant, and a portion (5 μl) of the supernatant was subjected to HPLC. HPLC was performed using a TSK-gel ODS-80Ts column (4.6 x 300 mm
Tosoh Corporation) was used, and 0.02 mol/L ammonium acetate buffer was used as the eluent.
(pH 4.0), with an elution temperature of 50°C and a flow rate of 0.5 ml/min. Product detection and quantification were performed using a fluorescence spectrophotometer FP-920 (Japan Bunko).
The assay was performed using a fluorochemical analyzer (manufactured by Hikari Co., Ltd.) (excitation wavelength: 320 nm, emission wavelength: 400 nm). The relative activity, with the activity when LNnT was used as the substrate being taken as 100%, is shown in Table 2.
Ta. When LNnT was used as a substrate, the conversion efficiency of the substrate to the product was 12.8%.
LNnT is a good substrate for G6 polypeptide (G6sec2), but LNT, L
It is clear that NFP-III and LNFP-V are hardly substrates.
In addition, the second GlcNAc residue from the non-reducing end in LNT
LNFP-II and LNDF are oligosaccharides in which fucose is attached via an α1,4 bond to
It was also revealed that H-II does not serve as a substrate for the G6 polypeptide. (3) Activity measurement using unlabeled oligosaccharides as substrates The glycosyltransferase reaction was performed as follows. 40 μl of assay solution [50 ml]
mol/l MOPS (pH 7.5), 5mmol/l UDP-GlcNAc
(manufactured by SIGMA), 5 mmol/l MnCl₂, 10 mmol/l sugar chain group
The mixture was incubated at 37°C for 16 hours in a purified enzyme solution (G6sec2).
After treating at 100°C for 5 minutes, the mixture was centrifuged at 10,000 x g for 20 minutes to obtain the supernatant.
A part of it is used for HPAE/PAD (High Performance Automated Electromagnetic Compatibility).
on Pulsed Amperometric Detection; DI
The analysis was carried out using a conventional method [Anal. Biochem.
m.,189, 151-162 (1990), J. Biol. Chem. ，27
3, 433-440 (1998) ]. As the purified enzyme (G6sec2), 10 μl of the enzyme obtained in (2) above was used.
As a substrate, unlabeled oligosaccharide (lactose (Ga
1β1-4Glc) and LNnT were used. For each substrate, an assay containing UDP-GlcNAc (sugar donor) was performed.
After reacting with the assay solution containing the solution and the non-solution, analysis was performed using HPAE/PAD.
The peak that appeared only in the assay solution containing UDP-GlcNAc was determined to be the product.
The product was identified based on whether its elution time matched that of the standard glycan.
Standard sugar chains include GlcNAcβ1-3Galβ1-4Glc and
GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Gl
c was used. The conversion efficiency to product when LNnT was used as a substrate was 0.48%, and when lactose was used as a substrate
When LNnT was used as a substrate, the conversion efficiency to the product was 0.30%.
When the activity was 100%, the relative activity when lactose was used as a substrate was 62.5%.
As a result, β1,3-N-acetylglucosaminyltransferase (G6)
It was found that lactose is also a good substrate for LNnT. (4) Activity measurement using glycolipids as substrates According to a known method [FEBS, 462, 289 (1999), J. Biol. Chem.
m.,269, 14730-14737 (1994), J. Biol. Chem
．267, 23507 (1992), J. Biol. Chem. ，267, 2
994 (1992)], the β1, β2, and β3 of G6 polypeptide were synthesized using glycolipid as a substrate.
3-N-acetylglucosaminyltransferase activity was measured.
Reaction solution [150 mmol/L sodium cacodylate (pH 7.2), 10 mm
ol/l UDP-GlcNAc (manufactured by SIGMA), 480 μmol/l U
DP-[¹⁴C]GlcNAc (Amersham), 0.4% Triro
n CF-54, 10 mmol/l MnCl₂, 250 μmol/l glycolipid,
The reaction was carried out at 37°C for 16 hours in purified enzyme (G6s
ec2) was used 10 μl of the enzyme obtained in (2) above.
The following are lactosylceramide, paraglobulin,
paragloboside, galactosylceramide
sylceramide) (type I), and galactosylceramide (gal
actosylceramide (type II) was used.
was obtained from Professor Yasunori Kushi of Tokyo Medical and Dental University. Other glycolipids were purchased from SIGMA.
After the reaction was completed, 200 μl of 0.1 mol/l KCl was added and the mixture was gently stirred.
After centrifugation, the supernatant was collected. After washing once with 10 ml of methanol,
Sep-Pak plus C18 equilibrated with 100 mol/L KCl twice
The supernatant was passed through Cartridge (manufactured by Waters) to separate glycolipids in the supernatant.
The cartridge was washed twice with 10 ml of pure water for HPLC.
After washing, the adsorbed glycolipids were eluted with 5 ml of methanol.
The liquid was concentrated to about 10 μl, and the concentrated liquid was applied to a TLC plate (HPTLC plate).
The sample was plotted on a silica gel 60 (manufactured by MERCK) and chloroform was added.
hum:methanol:water (0.2% CaCl₂(including) = 65:35:8 composition
The TLC plate was developed to a depth of 5 mm from the top edge.
After drying the plate, the plate was analyzed by a bio-image analyzer BAS2000 (Fuji Photo Film Co., Ltd.).
The amount of radiation incorporated into glycolipids was measured using a fluorochrome plated film (manufactured by Shin Film Co., Ltd.). FLAG-fused G6 polypeptide (G6sec2) reacts with lactosylceramide and
It was found that lactosylceramide and paragloboside are the substrates.
The conversion efficiencies to products when lagloboside was used as a substrate were 5.39% and 2.0%, respectively.
The G6 polypeptide when lactosylceramide was used as a substrate was 5.2%.
If the activity of the enzyme is 100%, the activity when paragloboside is used as a substrate is 4.
68%. On the other hand, G6 polypeptide (G6sec2) reacts with galactosylceramide (type
I) and galactosylceramide (type II).
. These results suggest that G6 polypeptide inhibits lactosylceramide and paraglobulin.
It is a β1,3-N-acetylglucosaminyltransferase that prefers α-glucosamine as a substrate.
By comparing with the results of Example 7 (2), it was found that secretory G6
(G6sec2) prefers paragloboside as a substrate over lactosylceramide.
It can be seen that the enzyme is
Certain β3GnTs arein vitroParagloboside is used as a substrate in
However, the activity is low when lactosylceramide is used as a substrate [Gly
cobiology,9, 1123 (1999)]. Therefore, G6 polypeptide
β1,3-N-acetylglucosamine thiol, which has different substrate specificity from β3GnT,
It is thought to be a transferase. In addition, G6 polypeptide and GlcNAcβ1,4-galactosyltransferase are
For example, lactosylceramide can be converted to paragloboside,
From neolactohexaosylceramide (Galβ1-4GlcNAcβ1-3
Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), and
can synthesize neolactohexaosylceramide from lactosylceramide.
Example 10: G6 polypeptide expression in cultured human cells transfected with an expression plasmid
Synthesis of glycolipids Namalwa cells (
10⁸cells), Namalwa cells (10⁸pieces),
Neutral glycolipids were extracted from Namalwa cells that had not been transfected with the plasmid.
Using immuno-TLC, N-
The composition and expression level of glycolipids containing acetyllactosamine structures were compared.
The method is based on a known method (Shujunsha Cell Engineering Special Issue Glycobiology Experimental Protocols
Anal. Biochem.,223, 232 (1994)].
The antibody recognizes the N-acetyllactosamine structure present at the non-reducing end of the sugar chain.
Monoclonal antibody capable of recognizing N-acetyllactosamine [anti-N-acetyllactosamine antibody 1B2-1B7
:Arch. Biochem. Biophysics. ，303, 125 (19
93), Infect. Immun. ，64, 4129 (1996), J. Co
mp. Neurol. ，22, 607 (1988)] antibody was used. 10⁷The neutral glycolipids were separated on a TLC plate (HPTLC plate Sil
The sample was spotted on a sheet of ica gel 60 (manufactured by MERCK) and then washed with chloroform:methanol.
Water (0.2% CaCl₂A developing solvent with a composition of 60:35:8 (including
As standard glycolipids, in the case of orcinol staining,
Glucosylceramide (2 μg; hereinafter, sometimes abbreviated as GlcCer),
Lactosylceramide (2 μg; hereinafter, sometimes abbreviated as LacCer),
Cetriaosylceramide (0.5 μg; hereinafter, Lc₃It is sometimes abbreviated as Cer
), and paragloboside (0.5 μg; hereafter referred to as nLc₄Abbreviated as Cer
), and paragloboside (nLc) in the case of immunohistochemistry.₄Cer) O
and neolactohexaosylceramide (hereinafter referred to as nLc₆It is sometimes abbreviated as Cer
Except for the standard glycolipids, two identical plates were prepared.
One plate was used for orcinol staining, and the other for immunostaining. After development, the plate was resuspended in isopropanol:water (0.2% CaCl₂Including): Meta
The plate was then immersed in a solution of 40:20:7 alcohol for 20 seconds.
The PVDF membrane (Immobilon: manufactured by Millipore) and glass
Covered with a microfiber filter (ATTO) and TLC Th
The glycolipids on the plate were analyzed using a Thermal Blotter (ATTO).
The conditions used were 180°C, level 8, 45 seconds, and transfer to a PVDF membrane.
The PVDF membrane was placed in a solution consisting of 5% skim milk and TBS-Tween 20.
After immersion for 1 hour, anti-N-acetyllactosamine antibody 1B2-1B7 (hybrid
5% skim milk containing 1/100 of the culture supernatant of rhesus maize, TBS-Tween
The PVDF membrane was immersed in a solution consisting of TBS-Tween 20 for 2 hours.
After washing three times, horseradish peroxidase-labeled anti-mouse IgM antibody (0
5% skim milk containing 4 μg/ml (manufactured by Jackson), TBS-Tw
The PVDF membrane was immersed in a solution of TBS-Tween 20 for 1 hour.
After washing three times with 20 ml of HCl, the plate was incubated with the ECL system (Amersham Pharmacia).
Antibody-bound glycolipids were detected using a ELISA kit (manufactured by the manufacturer). The results of orcinol staining are shown in Figure 4A, and the results of immunostaining are shown in Figure 4B.
Lane 1 of FIG. 4A shows standard glycolipids (GlcCer, LacCer, L
c₃Cer, nLc₄Cer), and lane 2 is the plasmid
Lane 3 is Namalwa cells not transfected with vector pAMo.
Lane 3 is Namalwa cells transfected with pAMo-G6.
Neutral glycolipids were extracted from each cell (lane 4) and developed on a TLC plate.
The results of orcinol staining are shown. Lane 1 in Figure 4B shows the standard
Glycolipids (nLc₄Cer, nLc₆Cer) and
Lane 2 is Namalwa cells without plasmid transfection, lane 3 is vector
Lane 3 is Namalwa cells transfected with pAMo, lane 4 is Namalwa cells transfected with pAMo-G6.
Neutral glycolipids were extracted from each of the Namalwa cells (lane 4) and analyzed by TLC.
After development into the glycan, the N-acetyllactosamine structure present at the non-reducing end of the glycan was
Antibody staining using a recognizable antibody (anti-N-acetyllactosamine antibody 1B2)
The arrow in B indicates the Namalw
a Glycolipids (nLc₄Cer and nLc₆Cer) position
In Namalwa cells expressing G6 polypeptide, the vector-introduced N
Compared to Namalwa cells and Namalwa cells without plasmid
Paraglycolipids, which have an N-acetyllactosamine structure at the non-reducing end of the sugar chain
Globoside (nLc₄Cer) and neolactohexaosylceramide (Gal
β1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1
-4Glc-ceramide: nLc₆The amount of ATP (abbreviated as Cer) increases.
It was revealed that the vector was introduced into Namalw
No difference was observed between the a cells and the Namalwa cells that had not been transfected with the plasmid.
(Figure 4B). These results and those of Example 9 suggest that G6 polypeptide acts as a N
-It is clear that it is involved in the synthesis of glycolipids with acetyllactosamine structure
As shown in Example 9, the G6 polypeptide produced lactosylceramide (
Galβ1-4Glc-ceramide) as a substrate to produce lactotriaosylceramide (
GlcNAcβ1-3Galβ1-4Glc-ceramide) can be synthesized.
Cells expressing β1,4-galactosyltransferase (e.g., Namalwa cells)
In the present study, lactotriaosylceramide synthesized by G6 polypeptide
is further converted to β1,4-galactose by the β1,4-galactosyltransferase.
By adding a bond, paragloboside (Galβ1-4GlcNAcβ
It is thought that ceramide is converted into 1-3Galβ1-4Glc-ceramide.
The results were obtained by injecting G6 polypeptide into appropriate cells (lactosylceramide, paragloboside, etc.).
The G6 polypeptide is expressed in a cell expressing a glycolipid that is a substrate for the G6 polypeptide.
This allows the galactose residues present at the non-reducing end of the glycolipid sugar chain to be used as the substrate.
It is possible to synthesize glycolipids to which N-acetylglucosamine is attached via a β1,3 bond.
Furthermore, the cells express the appropriate β1,4-galactosyltransferase.
In this case, the N-acetyllactosamine structure is located at the non-reducing end of the glycolipid sugar chain that serves as the substrate.
Alternatively, glycolipids with poly-N-acetyllactosamine sugar chains can be synthesized.
These results indicate that G6 polypeptide produces lactosylceramide β1,
It acts as a 3-N-acetylglucosaminyltransferase and acts to synthesize neolactoglycolipids and lactose.
It has also been shown that GlcNAcβ1,
By working in cooperation with 4-galactosyltransferase, the G6 polypeptide
Among them, poly-N-acetyllactosine such as neolactohexaosylceramide
It is also thought to be involved in the synthesis of glycolipids with amino sugar chains. The known enzyme (β3GnT) isin vitroIn lactosylceramide
It was shown that the enzyme exhibited weak β1,3-N-acetylglucosaminyltransferase activity against
However, whether lactosylceramide can actually be used as a substrate in cells is unclear.
Not shown. Example 11: Examination of the expression level of G6 gene transcription products in various cells G6 gene transcription products were quantified using a standard method [Proc. Natl. Acad. Sci.
i. USA,87, 2725 (1990), J. Biol. Chem. ，269 , 14730 (1994), JP 6-181759]
β-actin transcripts were also quantitatively quantified to correct gene expression levels.
This was performed using PCR. (1) Synthesis of single-stranded cDNA from various cells and cell lines The cell lines used were colon cancer cell lines (Colo201, Colo205, HCT-
15, SW480, SW620, WiDR, LS180), lung cancer cell line (AOI
, EBC-1, PC-1, A549, ABC-1, EHHA-9, HAL8, H
AL24, LX-1, PC-7, PC-9, PC-12, RERF-LC-MS
), gastric cancer cell lines (KATOIII, MKN1, MKN7, MKN28, MKN4
5, MKN74, TMK1, HSC43), neuroblastoma (NAGAI, NB-
9, SCCH-26, IMR32, SK-N-SH), glioblastoma cells
Strains (A172, KG-1-C, YKG-1, T98G, U251, U-118-
MG, G1-1), pancreatic cancer cell lines (Capan-1, Capan-2), prostate
Cancer cell line PC-3, liver cancer cell line HepG2, erythroleukemia cell type K562, granulocyte
/Monocytic cell line (HL-60, U-937, U266), T cell line Jurkat
, B cell line (Namalwa KJM-1, Namalwa, Daudi, Ra
Jurkat was obtained from the Aichi Cancer Center.
KATOIII and PC-9 were obtained from Immuno-Biological Laboratories.
Other cells are from the Japanese Collection of Research
ch Bioresources (JCRB) cell bank [Internet
Net address: http://cellbank.nihs.go.jp/) and
is an American Type Culture Collection
It is available from the Pe Culture Collection. It is also available from Nycomed Pharma (Nycomed Pharma) from peripheral blood of healthy adults.
Polymorphprep kit manufactured by Harma^TMPolymorphism
The mononuclear cells were isolated and obtained by the conventional method [J. Immunol.
．130, 706 (1983)], and further separated into monocytes and lymphocytes.
The total RNA of each cell was obtained by18, 5294 (19
The total RNA was prepared according to the following procedure. Single-stranded cDNA was synthesized from the total RNA using a kit (SU
PER^TMPreamplification System: manufactured by BRL)
For cell lines, 5 μg of total RNA was used to generate the IgG1-specific ...
First, single-stranded cDNA was synthesized from 1 μg of total RNA, and diluted 50 times with water.
The primers were oligonucleotides, and the resulting mixture was diluted 10-fold and used as a template for PCR.
(dT) primer was used. (2) Synthesis of single-stranded cDNA from various human tissues The same cDNA as in (1) above was synthesized from mRNA (Clontech) derived from various human organs.
Single-stranded cDNA was synthesized from 1 μg of mRNA.
The resulting mixture was diluted 240 times with water and used as a template for PCR.
The mRNA was extracted from the following 35 organs:
mRNA from 1 adrenal gland, 2 brain, 3 caudate nucleus, 4 hippocampus, 5 substantia nigra, and 6 thalamus was used.
, 7 kidney, 8 pancreas, 9 pituitary gland, 10 small intestine, 11 bone marrow, 12 amygdala, 13 cerebellum, 1
4 Corpus callosum, 15 Fetal brain, 16 Fetal kidney, 17 Fetal liver, 18 Fetal lung, 19 Heart, 20
Liver, 21 lung, 22 lymph node, 23 mammary gland, 24 placenta, 25 prostate, 26 salivary gland,
27 Skeletal muscle, 28 Spinal cord, 29 Spleen, 30 Stomach, 31 Testis, 32 Thymus, 33 Thyroid gland,
34 trachea, 35 uterus. Total RNA was also prepared from human colon tissue and analyzed in the same manner as for the cell line in (1) above.
Single-stranded cDNA was synthesized using 5 μg of total RNA as a template. (3) Preparation of standards and internal controls for quantitative PCR Standards and internal controls were prepared using pBS-G6 constructed in (5) of Example 3.
Controls were also prepared (see (a) and (b) below). Quantitation of β-actin transcripts was performed as previously reported [J. Biol. Chem.,269,
14730 (1994), JP-A-6-181759].
For quantification of transcriptional products, pUC119-ACT and pUC119-A
CTd is treated with a restriction enzyme (HindIII andAsp718)
After digestion and conversion to linear DNA, the standard and internal control
was used as a control [J. Biol. Chem.,269, 14730 (1994
After confirming that each plasmid was completely digested,
Yeast transfer RNA was used at 1 μg/ml in serial dilutions in water.
(a) Preparation of a standard for quantifying G6 transcripts The pBS-G6 gene, constructed in Example 3(5), was subjected to a control reaction to excise the G6 cDNA portion.
Restriction enzymes (EcoThe DNA was cleaved with RI and converted into linear DNA, which was then used as a quantification standard.
After confirming that the plasmid was completely digested, the yeast
The resulting product was serially diluted with water containing 1 μg/ml of transfer RNA. (b) Preparation of an internal control for quantification of G6 transcripts pBS-G6 was purified by restriction enzyme digestion.MscI andBglAfter cleavage with II, the linear DNA was
By ligating the G6 cDNA fragment with the 243 bp fragment, a 243 bp fragment was deleted from the G6 cDNA.
pBS-G6d was constructed. pBS-G6d was subjected to restriction cleavage to cut out the G6 cDNA portion.
enzyme(EcoThe DNA was cleaved with RI and converted into linear DNA, which was then used as an internal control for quantification.
After confirming that the plasmid was completely digested, the yeast was used as a vector.
The transfer RNA was serially diluted with water containing 1 μg/ml of transfer RNA and used. (4) Quantitation of G6 gene transcripts using quantitative PCR The single-stranded cDNAs derived from cell lines and normal tissues prepared in (1) and (2) above were used.
A quantitative PCR method, competitive PCR, was performed using A as a template.
As a PCR primer, the G6 transcript was detected using the primer represented by SEQ ID NO: 25.
CB513 having the base sequence and CB5 having the base sequence represented by SEQ ID NO: 28
15 was used. For detecting β-actin transcripts, the base sequence represented by SEQ ID NO: 29 was used.
CB53 having the base sequence represented by SEQ ID NO: 30 and CB54 having the base sequence represented by SEQ ID NO: 30 were used.
In addition, the same procedure was carried out using the standard and internal control prepared in (3) as templates.
A standard curve was created by performing PCR on 10 μl of the above single-stranded cDNA and 10 μl of the internal control plasmid.
(1 fg) in 50 μl of reaction solution [10 mmol/l Tris-HCl (
pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl₂,
0.2 mmol/l dNTP, 0.001% (w/v) gelatin, 0.2 μm
ol/l KCl gene-specific primer], DNA polymerase Ampli
Taq Gold^TM(Perkin Elmer) was added and PCR was performed.
The amount of internal control plasmid or standard plasmid
The amount of ATP was varied appropriately for each tissue and cell type. PCR was performed under the following conditions: For G6 transcript quantification, the PCR was performed at 95°C for 11 minutes, followed by 6 minutes at 95°C for 30 seconds.
42 cycles, each consisting of 1 minute at 0°C and 2 minutes at 72°C
When quantifying β-actin transcripts, the mixture was heated at 95°C for 11 minutes, followed by 1 minute at 95°C.
One cycle consisted of 1 minute at 65°C and 2 minutes at 72°C.
10 μl of the post-PCR solution was electrophoresed on a 1% agarose gel.
After staining with ethidium bromide, photographs were taken. The photographs were taken using the NIH Image System.
The intensity of staining of the amplified fragments was measured by scanning with
To quantify the transcripts more accurately, the number of PCR cycles was changed.
The amounts of the standard and internal control were adjusted according to the PCR conditions.
The cycle number was changed accordingly. Instead of cell-derived single-stranded cDNA, the standard prepared in (3) above was used.
PC using 125fg, 0.25fg, 0.5fg, 1fg, 2fg, and 4fg
R was performed, the amount of amplified fragment was measured, and the amount of cDNA and the amount of amplified fragment were plotted.
A calibration curve was created using the primers for quantifying the G6 transcript. When the primers for quantifying the G6 transcript were used, the G6 transcript and G6
A 458 bp DNA fragment was detected from the standard and a 458 bp DNA fragment was detected from the internal control of G6.
A 215 bp DNA fragment is amplified (see Figure 5). When the above primers for quantifying β-actin transcripts are used,
From the transcript and the β-actin standard, a 649 bp DNA fragment was detected.
-A 439 bp DNA fragment is amplified from the actin internal control (5th
(Photo of figure). The amount of G6 transcript is relative to the amount of β-actin transcript, which is set at 1000.
The values are shown in the histogram in Figure 5 and in Tables 3-1 and 3-2. The G6 transcript was expressed in most of the 36 human tissues examined (Figure 5).
The expression level varies depending on the tissue, including the cerebellum, pituitary gland, trachea, lung, colon, placenta, and testis.
It was also expressed relatively highly in monocytes and lymphocytes isolated from human peripheral blood.
G6 transcripts were also expressed in lymphocyte cells. In cell lines, colon cancer cell line Colo205, lung cancer cell lines (EBC1, H
AL8, LX-1, PC-7, RERF-LC-MS), gastric cancer cell lines (KATO III, MKN7, MKN28, MKN74, HSC43), neuroblastoma cell lines
Cell lines (NB9, SCCH-26, IMR32, SK-N-SH), glioblastoma
Tomatoma cell line (U251), pancreatic cancer cell line (Capan-1), B cell line (NA
LL-1) was relatively highly expressed (Tables 3-1 and 3-2). Example 12: Structural analysis of the chromosomal gene encoding G6 polypeptide Currently, the sequences of many human chromosomal genes with unknown functions are registered in databases.
Therefore, the sequence of the G6 cDNA of the present invention and the human G6 cDNAs registered in the database
By comparing the sequence of the G6 polypeptide of the present invention with that of the chromosomal gene,
The human chromosomal gene that encodes this gene (called the G6 chromosomal gene) was identified and its structure was clarified.
There is a possibility that the chromosomal gene sequence that matches the G6 cDNA sequence can be identified.
If it is registered, by comparing the cDNA sequence with the chromosomal gene sequence
the promoter region of the chromosomal gene encoding the polypeptide of the present invention,
The base sequence of human G6 cDNA (SEQ ID NO: 2) and the intron and intron structure can be determined.
National Center for Biotechnology on
National Center for Biological Information (NCBI) homepage (http://www
.ncbi.nlm.nih.gov/)
As a result of comparison with the sequence of the accession number AC025833 (published May 30, 2000),
A portion of the human chromosome working draft sequence (131,716 bp) [677
The assembly sequence consisting of the sequence from base 58 to base 72176 (assembl
y sequence): shown in SEQ ID NO: 30] is the same as the base sequence of G6 cDNA.
As a result of the analysis, it was found that the DNA had the base sequence shown in SEQ ID NO: 2.
The 1st to 3721st bases of the G6 cDNA sequence correspond to the 653rd base of SEQ ID NO: 30.
Therefore, the G6 cDNA shown in SEQ ID NO: 2 was identical to the 4373rd base from the first base.
It was revealed that the A portion is composed of one exon.
AATA present at positions 3702 to 3707 of the G6 cDNA sequence shown in
The sequence consisting of AA was considered to be a polyadenylation signal.
The upstream sequence (654 bp) of the G6 chromosomal gene is the promoter region (transcriptional control
The promoter region (654 bp) was considered to contain the sequence
Analysis software GENETYX-MAC 10.1 Transcription
Factor Database〔Nucleic Acids Research
ch,18, 1749 (1990), Trends in Biochemic
al Sciences,16, 455 (1991), Nucleic Aci.
ds Research,20S, 2091 (1992), Nucleic A
cids Research,21S, 3117 (1993)]
The binding sequences of transcription factors were identified using the Motif Search Program.
As a result of analyzing the presence of a consensus sequence of the promoter region,
It was determined that the sequence has the following sequence. Since the sequence of accession number AC025833 is derived from human chromosome 1,
The G6 chromosome gene was found to be located on human chromosome 1.
The location and structure of the gene on the chromosome (promoter region and exon region) are
Therefore, the structure of G6 cDNA and the function of the encoded polypeptide were clarified.
This was the first time that the species was identified.
The sequence encodes β1,3-N-acetylglucosaminyltransferase (G6 polypeptide).
It has not been clear until now that this will be done.Industrial Applicability The present invention provides a novel polymerase having β1,3-N-acetylglucosaminyltransferase activity.
polypeptide, a method for producing said polypeptide, DNA encoding said polypeptide,
A recombinant vector incorporating DNA, a transformant carrying the recombinant vector,
the recombinant polypeptide, an antibody that recognizes the polypeptide, and the polypeptide of the present invention using the antibody
Quantitative method and immunostaining method, GlcNAcβ1-3Gal using the polypeptide
sugar chains having the structure, poly-N-acetyllactosamine sugar chains and compounds containing the sugar chains
a method for producing a complex carbohydrate comprising the recombinant vector;
Sugar chains having NAcβ1-3Gal structures, poly-N-acetyllactosamine sugar chains
and a method for producing a glycoconjugate containing said sugar chain, and a gene encoding said polypeptide.
a method for screening a substance that alters the expression of the β1,3 polypeptide,
-Method for screening substances that alter N-acetylglucosaminyltransferase activity
The DNA or the antibody is used to treat inflammatory diseases and cancers (colon cancer, pancreatic cancer, stomach cancer, etc.)
a diagnostic method for the DNA, a method for altering expression of a gene encoding the polypeptide,
β1,3-N-acetylglucosaminyl transferase possessed by the substance or polypeptide
Inflammatory diseases and cancers (colon cancer, pancreatic cancer, stomach cancer, etc.) using substances that alter enzyme activity
) can be used to provide a treatment for the disease. "Sequence Listing Free Text" SEQ ID NO:4 - Synthetic DNA SEQ ID NO:5 - Synthetic DNA SEQ ID NO:6 - Synthetic DNA SEQ ID NO:7 - Synthetic DNA SEQ ID NO:8 - Synthetic DNA SEQ ID NO:9 - Synthetic DNA SEQ ID NO:10 - Synthetic DNA SEQ ID NO:11 - Synthetic DNA SEQ ID NO:12 - Synthetic DNA SEQ ID NO:13 - Amino acid sequence of FLAG peptide SEQ ID NO:14 - Synthetic DNA SEQ ID NO:15 - Synthetic DNA SEQ ID NO:16 - Synthetic DNA SEQ ID NO:17 - Synthetic DNA SEQ ID NO:18 - Synthetic DNA SEQ ID NO:19 - Synthetic DNA SEQ ID NO:20 - Synthetic DNA SEQ ID NO:21 - Synthetic DNA SEQ ID NO:22 - Synthetic DNA SEQ ID NO:23 - Synthetic DNA SEQ ID NO:24 - Synthetic DNA SEQ ID NO:25 - Synthetic DNA SEQ ID NO:26 - Synthetic DNA SEQ ID NO:28 - Synthetic DNA SEQ ID NO:29 - Synthetic DNA SEQ ID NO:31 - Synthetic DNA

[Sequence List]

[Brief explanation of the drawings]

Figure 1 shows the results of FACS analysis of the expression level of poly-N-acetyllactosamine sugar chains in HCT-15 cells transfected with a G6 polypeptide expression plasmid. Figure 2 shows the results of FACS analysis of the expression level of poly-N-acetyllactosamine sugar chains in HCT-15 cells transfected with a G6 polypeptide expression plasmid, after treating them with various sugar chain synthesis inhibitors. This analysis examined whether G6 polypeptide is involved in the synthesis of glycoprotein sugar chains or glycolipid sugar chains. Figure 3 shows the results of silver staining after SDS-polyacrylamide gel electrophoresis of purified FLAG peptide-fused secreted G6 polypeptide produced in insect cells. Figure B shows the results of Western blotting using an anti-FLAG peptide antibody after SDS-polyacrylamide gel electrophoresis of purified FLAG peptide-fused secreted G6 polypeptide produced in insect cells. Figure 4 shows the results of orcinol staining after extracting neutral glycolipids from Namalwa cells into which an expression plasmid for G6 polypeptide has been introduced, and then spreading them on a TLC plate.
4 shows the results of antibody staining using anti-N-acetyllactosamine antibody 1B2 after neutral glycolipids extracted from malwa cells were developed on a TLC plate. 5 shows electrophoresis results obtained by examining the expression levels of G6 transcripts and β-actin transcripts in 36 types of human organs using competitive PCR. The histogram is a graph showing the expression level of G6 transcripts when the expression level of β-actin is set to 1,000.

─────────────────────────────────────────────────────── Continued from the front page (51) Int.Cl. ⁷ Identification Symbol FI A61K 45/00 A61K 48/00 48/00 A61P 29/00 A61P 29/00 35/00 35/00 35/04 35/04 43/00 111 43/00 111 C07K 16/40 C07K 16/40 C12N 1/21 C12N 1/21 9/10 5/10 C12P 19/26 9/10 C12Q 1/02 C12P 19/26 1/48 Z C12Q 1/02 1/68 A 1/48 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 C12N 5/00 B 33/50 C A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG), AP (G H, GM, KE, LS, MW, MZ, SD, SL, SZ , TZ, UG, ZW), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, B Y, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, I L, IN, IS, JP, KE, KG, KR, KZ, LC , LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, P H, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor: Hiroyuki Mio, Kyowa Hakko Kogyo Co., Ltd., Head Office, 1-6-1 Otemachi, Chiyoda-ku, Tokyo (72) Inventor: Satoshi Nakagawa, Kyowa Hakko Kogyo Co., Ltd., Tokyo Research Laboratory, 3-6-6 Asahimachi, Machida-shi, Tokyo (72) Inventor: Susumu Sekine, Kyowa Hakko Kogyo Co., Ltd., Tokyo Research Laboratory, 3-6-6 Asahimachi, Machida-shi, Tokyo (72) Inventor: Akira Togayanai, 1-11-42 Shimosato, Higashikurume-shi, Tokyo (Note) This publication is based on the gazette published internationally by the International Bureau of Patent Publication (WIPO). The effect of the international publication of the Japanese patent application (Japanese utility model registration application) related to this publication arises pursuant to Article 184-10, Paragraph 1 of the Patent Act (Article 48-13, Paragraph 2 of the Utility Model Act), and is unrelated to this publication.

Claims (79)

[Claims] 1. A polypeptide consisting of the amino acid sequence represented by SEQ ID NO:1. 2. A polypeptide comprising the amino acid sequence from the 39th to the 378th amino acids of the amino acid sequence represented by SEQ ID NO:1. 3. A polypeptide comprising the amino acid sequence of the polypeptide according to claim 1 or 2, in which one or more amino acids have been deleted, substituted or added, and having β1,3-N-acetylglucosaminyltransferase activity. 4. A polypeptide comprising the amino acid sequence of claim 1 or 2 and 6
A polypeptide comprising an amino acid sequence having 0% or more homology to the β1,3-N-acetylglucosaminyltransferase activity. 5. A polypeptide according to any one of claims 3 and 4, wherein the β1,3-N-acetylglucosaminyltransferase activity is the activity of transferring N-acetylglucosamine via a β1,3 bond to a galactose residue present at the non-reducing end of a sugar chain. 6. The method of claim 1, wherein the β1,3-N-acetylglucosaminyltransferase activity is i) galactose, N-acetyllactosamine (Galβ1-4GlcNAc), Gal
β1-3GlcNAc or lactose (Galβ1-4Glc), ii) oligosaccharides having a galactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure at the non-reducing end, and iii) galactose, N-
A polypeptide described in any one of claims 3 to 5, which has the activity of transferring N-acetylglucosamine via a β1,3 bond to a galactose residue present at the non-reducing end of the sugar chain of an acceptor substrate selected from complex carbohydrates having an acetyllactosamine, Galβ1-3GlcNAc or lactose structure at the non-reducing end. 7. Galactose, N-acetyllactosamine, Galβ1-3Gl
The glycoconjugate having a cNAc or lactose structure at the non-reducing end is lactosylceramide (Galβ1-4Glc-ceramide) or paragloboside (Galβ1
The polypeptide of claim 6, which is β1-4GlcNAcβ1-3Galβ1-4Glc-ceramide. 8. The polypeptide according to claim 6 or 7, wherein the complex carbohydrate is a complex carbohydrate selected from glycoproteins, glycolipids, proteoglycans, glycopeptides, lipopolysaccharides, peptidoglycans and glycosides in which a sugar chain is bound to a steroid compound. 9. A sugar chain synthesizing agent comprising the polypeptide according to any one of claims 1 to 8 as an active ingredient. 10. A DNA encoding the polypeptide according to any one of claims 1 to 8. 11. DNA having the base sequence shown in SEQ ID NO: 2. 12. A DNA having the base sequence from bases 135 to 1268 of the base sequence represented by SEQ ID NO: 2. 13. A DNA having the base sequence from base 249 to base 1268 of the base sequence represented by SEQ ID NO: 2. [Claim 14] A DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence of the DNA described in any one of claims 10 to 13 and encodes a polypeptide having β1,3-N-acetylglucosaminyltransferase activity. 15. The DNA according to claim 14, wherein the β1,3-N-acetylglucosaminyltransferase activity is the activity of transferring N-acetylglucosamine via a β1,3 bond to a galactose residue present at the non-reducing end of a sugar chain. 16. A method for producing a β1,3-N-acetylglucosaminyltransferase comprising the steps of: i) galactose, N-acetyllactosamine (Galβ1-4GlcNAc), Ga
ii) oligosaccharides having a galactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure at the non-reducing end, and iii) galactose, N
A DNA as described in claim 14 or 15, which has the activity of transferring N-acetylglucosamine via a β1,3 bond to a galactose residue present at the non-reducing end of the sugar chain of an acceptor substrate selected from complex carbohydrates having an N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure at the non-reducing end. 17. Galactose, N-acetyllactosamine, Galβ1-3G
The DNA according to claim 16, wherein the complex carbohydrate having an lcNAc or lactose structure at the non-reducing end is lactosylceramide or paragloboside. 18. The DNA according to claim 16 or 17, wherein the complex carbohydrate is a complex carbohydrate selected from glycoproteins, glycolipids, proteoglycans, glycopeptides, lipopolysaccharides, peptidoglycans and glycosides in which a sugar chain is bound to a steroid compound. 19. A DNA having a sequence complementary to the base sequence of the DNA according to any one of claims 10 to 18. 20. A recombinant vector obtained by incorporating the DNA according to any one of claims 10 to 18 into a vector. 21. A recombinant vector obtained by incorporating RNA having a sequence homologous to the DNA according to any one of claims 10 to 18 into a vector. 22. A transformant carrying the recombinant vector according to claim 20 or 21. 23. The transformant according to claim 22, which is a transformant selected from the group consisting of a microorganism, an animal cell, a plant cell, and an insect cell. 24. The method according to claim 24, wherein the microorganism is a microorganism belonging to the genus Escherichia .
The transformant according to claim 23. 25. The transformant according to claim 23, wherein the animal cell is an animal cell selected from the group consisting of mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CHO cells, BHK cells, African green monkey kidney cells, Namalwa cells, Namalwa KJM-1 cells, human embryonic kidney cells and human leukemia cells. 26. The plant cell is selected from the group consisting of tobacco, potato, tomato, carrot, soybean,
24. The transformant according to claim 23, which is a plant cell selected from the group consisting of plant cells of rapeseed, alfalfa, rice, wheat, barley, rye, corn, or flax. 27. The transformant according to claim 23, wherein the insect cell is an insect cell selected from the group consisting of an ovary cell of Spodoptera frugiperda , an ovary cell of Trichoplusia ni , and an ovary cell of a silkworm. 28. The transformant according to claim 22, which is a non-human transgenic animal or a transgenic plant. 29. A method for producing a polypeptide, comprising culturing a transformant according to any one of claims 22 to 27 in a medium, producing and accumulating the polypeptide according to any one of claims 1 to 8 in the culture, and recovering the polypeptide from the culture. 30. A method of producing and accumulating a polypeptide according to any one of claims 1 to 8 in a non-human transgenic animal according to claim 28,
A method for producing said polypeptide, which comprises collecting said polypeptide from said animal. 31. The method of claim 30, wherein the accumulation is in the milk of the animal. 32. Cultivating the transgenic plant of claim 28 and producing the plant of claim 1.
9. A method for producing the polypeptide according to any one of claims 1 to 8, which comprises producing and accumulating the polypeptide in the plant, and collecting the polypeptide from the plant. 33. A method for producing a polypeptide according to any one of claims 1 to 8, comprising synthesizing the polypeptide according to any one of claims 1 to 8 using the DNA according to any one of claims 10 to 18 in an in vitro transcription/translation system. 34. A method for producing a sugar chain synthesizing agent according to claim 9, comprising the steps of: (a) the enzyme source; (b) i) lactosylceramide (Galβ1-4Glc-ceramide) or paragloboside (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide); ii) galactose, N-acetyllactosamine (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide);
cNAc), Galβ1-3GlcNAc or lactose (Galβ1-4G
lc), iii) galactose, N-acetyllactosamine (Galβ1-4G
lcNAc), Galβ1-3GlcNAc or lactose (Galβ1-4
Glc) oligosaccharides having a structure at the non-reducing end, and iv) galactose, N-
an acceptor substrate selected from a complex carbohydrate having an acetyllactosamine, Galβ1-3GlcNAc, or lactose structure at the non-reducing end, and (c) uridine-5′-diphosphate N-acetylglucosamine (UDP-GlcNAc),
Ac) in an aqueous medium, producing and accumulating in the aqueous medium a sugar chain or a glycoconjugate in which N-acetylglucosamine is attached to a galactose residue of the acceptor substrate via a β1,3 bond, and collecting the sugar chain or glycoconjugate from the aqueous medium. [Claim 35] A method for producing a sugar chain or glycoconjugate having galactose attached thereto, comprising using as an acceptor substrate the sugar chain or glycoconjugate having N-acetylglucosamine attached thereto obtained by the method of claim 34, placing (a) the acceptor substrate, (b) GlcNAcβ1,4-galactosyltransferase, and (c) uridine-5'-diphosphate galactose (UDP-Gal) in an aqueous medium, producing and accumulating in the aqueous medium a reaction product in which galactose is attached via a β1,4 bond to the N-acetylglucosamine residue at the non-reducing end of the acceptor substrate, and collecting the sugar chain or glycoconjugate having galactose attached thereto from the aqueous medium. 36. A method for producing a sugar chain synthesizing enzyme comprising using the sugar chain synthesizing agent according to claim 9 as an enzyme source, and (a) the enzyme source, (b) GlcNAcβ1,4-galactosyltransferase, and (c) i) lactosylceramide (Galβ1-4Glc-ceramide) or paragloboside (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), ii) galactose, N-acetyllactosamine (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), and
cNAc), Galβ1-3GlcNAc or lactose (Galβ1-4G
lc), iii) galactose, N-acetyllactosamine (Galβ1-4G
lcNAc), Galβ1-3GlcNAc or lactose (Galβ1-4
(iv) an oligosaccharide having a galactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure at its non-reducing end; and (v) a sugar chain or a glycoconjugate obtained by the method according to claim 33 or 34.
a method for producing a sugar chain or a glycoconjugate having a poly-N-acetyllactosamine sugar chain attached thereto, the method comprising the steps of: (a) introducing (a) uridine-5'-diphosphate galactose (UDP-Gal) into an aqueous medium; (b) allowing a reaction product having a poly-N-acetyllactosamine sugar chain attached to the non-reducing end of the acceptor substrate to be produced and accumulated in the aqueous medium; and (c) collecting the sugar chain or glycoconjugate having the poly-N-acetyllactosamine sugar chain attached thereto from the aqueous medium. 37. A method for producing a strain of a plant using the transformant according to any one of claims 22 to 27,
GlcNAcβ1-3Galβ1-4Glc-ceramide, lacto-series glycolipid (Ga
glycolipids having 1β1-3GlcNAcβ1-3Galβ1-4Glc-ceramide as the backbone), neolacto-series glycolipids (Galβ1-4GlcNAcβ1-3G
alβ1-4Glc-ceramide as a glycolipid), GlcNAcβ1
-Sugars with 3Gal structure, GlcNAcβ1-3Galβ1-4GlcNAc
sugars having a GlcNAcβ1-3Galβ1-3GlcNAc structure, sugars having a GlcNAcβ1-3Galβ1-4Glc structure, (Galβ
1-4GlcNAcβ1-3) _n Galβ1-4GlcNAc structure, n is 1
and (Galβ1-4GlcNAcβ1-3) _n Galβ1-
A method for producing a sugar chain or a complex carbohydrate, comprising producing and accumulating a sugar chain consisting of a sugar selected from the group consisting of sugars having a 4Glc structure and n being 1 or greater, or a complex carbohydrate containing said sugar chain, and collecting said sugar chain or complex carbohydrate from the culture. 38. A method for producing GlcNAcβ1-3Galβ1-4Glc-ceramide, lacto-series glycolipid (Galβ1-3GlcNAcβ1-3Galβ1-4Glc) using the non-human transgenic animal or transgenic plant according to claim 28.
glycolipids having c-ceramide as the backbone), neolacto-series glycolipids (Galβ1-
Glycolipids having a GlcNAcβ1-3Galβ1-4Glc-ceramide backbone, sugars having a GlcNAcβ1-3Gal structure, GlcNAcβ1-3G
Sugars having alβ1-4GlcNAc structure, GlcNAcβ1-3Galβ1-
Sugars having a GlcNAcβ1-3Galβ1-4Glc structure, sugars having a GlcNAcβ1-3Galβ1-4Glc structure, (Galβ1-4GlcNAcβ1-3) _nGalβ1-4Gl
Sugars having a cNAc structure and n being 1 or greater, and (Galβ1-4GlcNAc
A method for producing a sugar chain or a glycoconjugate, comprising producing and accumulating a sugar chain consisting of a sugar selected from the group consisting of sugars having a β1-3) _nGalβ1-4Glc structure, wherein n is 1 or more, or a glycoconjugate containing said sugar chain, and collecting said sugar chain or glycoconjugate from said individual. [Claim 39] A production method described in any one of claims 34 to 38, wherein the complex carbohydrate is a complex carbohydrate selected from glycoproteins, glycolipids, proteoglycans, glycopeptides, lipopolysaccharides, peptidoglycans, and glycosides in which a sugar chain is bound to a steroid compound. 40. The method of claim 38, wherein the accumulation is in the milk of the animal. 41. An oligonucleotide having the same sequence as 5 to 120 consecutive bases of the base sequence of the DNA according to any one of claims 10 to 19, and an oligonucleotide which is a derivative of said oligonucleotide. [Claim 42] A method for quantifying the expression level of a gene encoding a polypeptide described in any one of claims 1 to 8 by a hybridization method using a DNA described in any one of claims 10 to 19, a partial fragment of said DNA, or an oligonucleotide described in claim 41. 43. A polymerase using the oligonucleotide according to claim 41.
A method for quantifying the expression level of a gene encoding the polypeptide according to any one of claims 1 to 8 by chain reaction. 44. A method for detecting inflammation, cancer or cancer metastasis, using the method according to claim 42 or 43. 45. A detection agent for inflammation, cancer or cancer metastasis, comprising the DNA according to any one of claims 10 to 19, a partial fragment of said DNA or the oligonucleotide according to claim 41. 46. A method for diagnosing a functional abnormality of a gene encoding the polypeptide according to any one of claims 1 to 8, by detecting a mutation in said gene. [Claim 47] A method for detecting a mutation in a gene encoding a polypeptide described in any one of claims 1 to 8 by a hybridization method using a DNA described in any one of claims 10 to 19, a partial fragment of said DNA, or an oligonucleotide described in claim 41. 48. A polymerase using the oligonucleotide according to claim 41.
A method for detecting a mutation in a gene encoding the polypeptide according to any one of claims 1 to 8 by chain reaction. 49. A method for inhibiting transcription of a gene encoding a polypeptide according to any one of claims 1 to 8 or translation of mRNA, using the oligonucleotide according to claim 41. 50. An antibody that recognizes the polypeptide described in any one of claims 1 to 8. 51. An immunological method for detecting a polypeptide according to any one of claims 1 to 8, using the antibody according to claim 50. 52. An immunohistochemical staining method, comprising detecting the polypeptide according to any one of claims 1 to 8 using the antibody according to claim 50. 53. An immunohistochemical staining agent comprising the antibody according to claim 50. 54. A pharmaceutical comprising the polypeptide according to any one of claims 1 to 8. 55. The pharmaceutical composition according to claim 54, which is used for the treatment, prevention and/or diagnosis of inflammatory diseases, cancer or cancer metastasis. 56. A medicine comprising the DNA according to any one of claims 10 to 19, a partial fragment of said DNA, or the oligonucleotide according to claim 41. 57. The pharmaceutical composition according to claim 56, which is used for the treatment, prevention and/or diagnosis of inflammatory diseases, cancer or cancer metastasis. 58. A pharmaceutical comprising the recombinant vector according to claim 20 or 21. 59. The pharmaceutical composition according to claim 58, which is used for the treatment, prevention and/or diagnosis of inflammatory diseases, cancer or cancer metastasis. 60. A pharmaceutical comprising the antibody according to claim 50. 61. The pharmaceutical composition according to claim 60, which is used for the treatment, prevention and/or diagnosis of inflammatory diseases, cancer or cancer metastasis. [Claim 62] A method for screening a compound that alters the β1,3-N-acetylglucosaminyltransferase activity of a polypeptide, comprising contacting a polypeptide described in any one of claims 1 to 8 with a test sample and measuring the alteration in the β1,3-N-acetylglucosaminyltransferase activity of the polypeptide due to the test sample. 63. The screening method according to claim 62, wherein the β1,3-N-acetylglucosaminyltransferase activity is the activity of transferring N-acetylglucosamine via a β1,3 bond to a galactose residue present at the non-reducing end of a sugar chain. 64. The method of claim 64, wherein the β1,3-N-acetylglucosaminyltransferase activity is i) galactose, N-acetyllactosamine (Galβ1-4GlcNAc), Ga
1β1-3GlcNAc or lactose (Galβ1-4G
lc), ii) Galactose, N-acetyllactosamine, Galβ1-3Gl
an oligosaccharide having a cNAc or lactose structure at the non-reducing end, and iii)
A screening method according to claim 62 or 63, which is the activity of transferring N-acetylglucosamine via a β1,3 bond to a galactose residue present at the non-reducing end of the sugar chain of an acceptor substrate selected from complex carbohydrates having a galactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure at the non-reducing end. 65. Galactose, N-acetyllactosamine, Galβ1-3G
The screening method according to claim 64, wherein the complex carbohydrate having an lcNAc or lactose structure at the non-reducing end is lactosylceramide or paragloboside. 66. The screening method according to claim 64 or 65, wherein the complex carbohydrate is a complex carbohydrate selected from glycoproteins, glycolipids, proteoglycans, glycopeptides, lipopolysaccharides, peptidoglycans and glycosides in which a sugar chain is bound to a steroid compound. 67. A method for producing a cell expressing the polypeptide according to any one of claims 1 to 8, comprising contacting the cell with a test sample, and detecting an antibody that recognizes paragloboside or poly-N
A method for screening for compounds that alter the expression of genes encoding said polypeptides, characterized by quantifying paragloboside or poly-N-acetyllactosamine sugar chains using at least one selected from the group consisting of antibodies and lectins that recognize said acetyllactosamine sugar chains. [Claim 68] A method for screening a compound that alters the expression of a gene encoding a polypeptide described in claims 1 to 8, comprising contacting a test sample with a cell that expresses the polypeptide and quantifying the polypeptide using the antibody described in claim 50. 69. A promoter DNA that controls the transcription of a gene encoding the polypeptide according to any one of claims 1 to 8. 70. The promoter DNA is capable of targeting white blood cells, nerve cells, tracheal cells,
Lung cells, colon cells, placental cells, neuroblastoma cells, glioblastoma cells,
The promoter DNA of claim 69, which is a promoter that functions in cells selected from colon cancer cells, lung cancer cells, pancreatic cancer cells, gastric cancer cells and leukemia cells. 71. The promoter DNA according to claim 69 or 70, which is derived from a human, a rat, or a mouse. 72. The promoter DNA according to any one of claims 69 to 71.
and a method for screening a compound that alters the efficiency of transcription by the promoter, which comprises transforming an animal cell with a plasmid containing a reporter gene linked downstream of the promoter DNA, contacting the transformant with a test sample, and quantifying the translation product of the reporter gene. 73. The reporter gene, comprising a chloramphenicol acetyltransferase gene, a β-galactosidase gene, a β-lactamase gene,
73. The method of claim 72, wherein the gene is selected from the group consisting of a luciferase gene and a green fluorescent protein gene. 74. A compound obtained by the screening method according to any one of claims 62 to 68, 72 and 73. 75. A non-human knockout animal in which the DNA encoding the polypeptide according to any one of claims 1 to 8 is deleted or mutated. 76. The knockout animal of claim 75, wherein the non-human knockout animal is a mouse. [Claim 77] A method for controlling cell differentiation, mutual recognition, and migration, characterized by introducing DNA described in claims 10 to 18 or RNA consisting of a sequence homologous to said DNA, or a recombinant vector described in claim 20 or 21 into cells and expressing a polypeptide described in claims 1 to 8. 78. The method according to claim 77, wherein the cells are selected from the group consisting of blood cells, nerve cells, stem cells, and cancer cells. [Claim 79] A method for promoting differentiation of promyelocytic cells into granulocytic cells, characterized by introducing DNA described in claims 10 to 18 or RNA consisting of a sequence homologous to said DNA, or a recombinant vector described in claim 20 or 21 into promyelocytic cells to express the polypeptide described in claims 1 to 8.