JP2004194675A - Novel glycosyltransferase, gene for encoding same and method for producing same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel glycosyltransferase, β1,6-N-acetylglucosaminyltransferase. <P>SOLUTION: Disclosed is a β1,6-N-acetylglucosaminyltransferase having the following enzymatic characteristics: an action for transferring N-acetylglucosamine from UDP-N-acetylglucosamine to α-6-D-mannoside; and a molecular weight of about 73,000 (determined by SDS-PAGE in the absence of a reducing agent) as well as about 73,000 and about 60,000 (determined by SDS-PAGE in the presence of a reducing agent). <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to an enzyme that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to α-6-D-mannoside (UDP-N-acetylglucosamine: α-6-D-mannoside, β1,6-N-acetyl). Glucosaminyltransferase (hereinafter abbreviated as GnT-V), a gene system encoding the same, and a method for producing GnT-V.

  Asparagine-type sugar chains (Asn-type sugar chains) found in glycoproteins are classified into three types: high mannose type, complex type and mixed type, based on their constituent sugars and branched types. First, the biosynthesis of these Asn-type sugar chains starts from the collective transfer of the sugar chain portion from the lipid intermediate to the asparagine of the polypeptide chain during translation on the lumen side of the rough endoplasmic reticulum.

  After that, glucose and some mannose are removed from the rough endoplasmic reticulum, but some glycoproteins with Asn-type sugar chains localized in the rough endoplasmic reticulum remain as they are. It becomes. Other organelle glycoproteins, cell surface glycoproteins or secreted glycoproteins are transferred to the Golgi body by vesicle transport, and mannose is further removed. In this Golgi apparatus, N-acetylglucosamine is introduced by the action of the N-acetylglucosaminyltransferase group, which is a Golgi enzyme, and takes a branched structure. Due to the formation of this branched structure, conversion of high-mannose-type sugar chains to hybrid-type and complex-type sugar chains is started, fucose is introduced, and galactose is introduced in the trans-Golgi region. The acid is introduced to complete the biosynthesis of the Asn-type sugar chain.

  It is known that various enzymes work as catalysts at each step of the series of Asn-type sugar chain synthesis. Among them, six types of N-acetylglucosaminyltransferases are known as enzymes that catalyze the transfer introduction reaction of N-acetylglucosamine in the formation of various branched structures of Asn-type sugar chains. Schachter et al. (Brockhausen, I., Caarver, J., and Schachter, H., Biochem. Cell Biol., 66, 1134 (1988)) describe Manα1-3 (Manα1-6) Manβ1-4GlcNAcβ1-4GlcNAc. These six enzymes that transfer N-acetylglucosamine to the core structure of the structure were named GnT-I to GnT-VI.

  Among them, GnT-V is an enzyme involved in the formation of a β (1,6) branch structure (-[GlcNAc-β (1,6) Man-α (1,6) Man]-). It is known that the β (1,6) branch structure is remarkably increased in transformed cells and tumorigenic cells (Pierce, M., Arango, J., Tahir, SH). , And Hindsgaul, O., Biochem.Biophys.Res.Commun., 146,679-684 (1987) and Arango, J., and Piercece, M., J. Cell.Biochem., 257, 13421-13427 (1987). 1982)).

  It has also been shown that there is a relationship between the metastatic potential of tumorigenic cells and the appearance of β (1,6) branch (Hiraizumi, S., Takasaki, S., Shiroki, K., Kochibe). , N., and Kobata, A., Arch. Biochem. Biophys. 280, 9-19, (1990)). In humans, it has been reported that the expression of β (1,6) branch is increased in 50% of breast cancer biopsy cases (Dennis, JW, and Laferte, S. Cancer Res. 49, 945-950, (1989)).

  In any case, it has been found that the appearance of the β (1,6) branch structure is accompanied by an increase in GnT-V activity. Thus, GnT-V is not only important in catalyzing the formation of the β (1,6) branch structure in the sugar chain biosynthesis pathway, but also related to the metastasis ability and malignancy of cancer cells. In this respect, it is also an important key enzyme.

  For GnT-I among these six N-acetylglucosaminyltransferases, the human and rabbit cDNA structures were revealed (Kumar, R., Yang, J., Larsen, RD, and Stanley). Natl.Acad.Sci.U.S.A. 87, 9948-9952, (1990) and Sarkar, M., Hull, E., Nishikawa, Y., Simpson, R.J., Moritz. , RL, Dunn, R., and Schachter, H., Proc.Natl.Acad.Sci.U.S.A. 88, 234-238, (1991)).

  On the other hand, the presence of GnT-V is presumed from the enzyme activity, and it has been attempted to purify GnT-V from various tissues in a living body. However, when the enzyme is solubilized, it has an extremely unstable property. Therefore, purification was extremely difficult (Nishikawa, A., Gu, J., Fujiji, S., and Taniguchi, N. Biochim. Biophys. Acta 1035, 313-318, (1990)). However, recently, an example in which GnT-V was isolated and purified from rat kidney was reported (Shoreibah, M., G., Hindgaul, O., and Pierce, M., J. Biol. Chem. 267, 2920-2927, (1992)).

  However, detailed information on the gene of the enzyme has not been obtained from purified rat GnT-V. Furthermore, no information has been obtained on human GnT-V, including its enzymatic properties. GnT-V is not only very useful in diagnosis as a marker of malignancy of cancerous cells, but also a cancer metastasis inhibitor by establishing a screening system for a specific inhibitor of the present enzyme using the present enzyme. Since it is possible for the first time to design GnT-V, there is a demand for a method for isolating and purifying human-derived GnT-V and for analyzing its structure including genes and mass-producing it.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide human-derived GnT-V. Further, the present invention is to isolate a gene related to the enzyme, develop a method for mass-producing the enzyme using the gene, and to enable uniform production of sugar chains in production of useful substances by genetic manipulation. The purpose is.

  The present inventors have focused on the culture supernatant of cancer cells as a starting material for purifying the enzyme. However, the present inventors considered that it was difficult to purify a culture supernatant of cancer cells that could not grow without adding bovine serum or the like. Accordingly, the present inventors have conducted intensive studies to adapt various cancer cells to a protein-free medium, and succeeded in establishing cancer cells adapted to tens of types of protein-free medium. Among them, the present enzyme activity was observed in the culture supernatant of a protein-free medium of QG cells derived from human lung cancer (small cell carcinoma), and the enzyme was successfully purified.

Accordingly, the present invention provides the following enzymatic properties:
(1) action; transferring N-acetylglucosamine from UDP-N-acetylglucosamine to α-6-D-mannoside;
(2) Substrate specificity: When GnGn-bi-PA is defined as a receptor, 100 is used, and about 78% for GnGnF-bi-PA, about 125% for GnGnGn-tri-PA, and about 66% for GnM-PA. % Reactivity;
(3) optimum pH; 6.2-6.3;
(4) inhibition, activation and stabilization; does not require Mn ²⁺ for expression of activity, and does not inhibit activity in the presence of 20 mM EDTA;

(5) molecular weight; about 73,000 (by SDS-PAGE in the absence of a reducing agent) and about 73,000 and about 60,000 (by SDS-PAGE in the presence of a reducing agent);
(6) Km values; Km values for acceptor GnGn-bi-PA and donor UDP-GlcNAc are 133 μM, 3.5 mM, respectively; and (7) with the following peptide fragments:
(1) Thr-Pro-Trp-Gly-Lys
(2) Asn-Ile-Pro-Ser-Tyr-Val
(3) Val-Leu-Asp-Ser-Phe-Gly-Thr-Glu-Pro-Glu-Phe-Asn-His-Ala-Asn-Tyr-Ala
(4) Asp-Leu-Gln-Phe-Leu-Leu,
(5) Asn-Thr-Asp-Phe-Phe-Ile-Gly
And β1,6-N-acetylglucosaminyltransferase having the formula:

The β1,6-N-acetylglucosaminyltransferase of the present invention is, for example, an amino acid sequence comprising at least the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence in which one or more amino acids are modified in the amino acid sequence Having. Here, amino acid modification means that an amino acid has been added, removed and / or substituted.
The present invention further provides a method for producing the enzyme, which comprises culturing QG cells or human colon cancer cells CL0201 cells derived from human lung cancer, and collecting the enzyme from the culture.

The present invention further provides the method for producing a β1,6-N-acetylglucosaminyltransferase enzyme, wherein a host transformed with an expression vector containing a DNA encoding the enzyme is cultured or bred, and the host is transformed from the host. A method is provided wherein the enzyme is collected.
The invention further relates to the gene systems encoding said enzymes, ie DNA, expression vectors, and transformed hosts.

  QG cells derived from human lung cancer (small cell carcinoma) can be cultured by any method commonly used for culturing animal cells. It is also possible to perform stationary culture using a medium containing no protein. A specific example is described in Example 1. The human lung cancer (small cell carcinoma) -derived QG cells were named Human lung carcinoma SBM331, and deposited on August 18, 1992 with the Research Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology. No. (FERM BP-3967) under international deposit under the Budapest Treaty.

  The enzyme activity can be easily measured by labeling a receptor with a radioisotope or a fluorescent substance, reacting the enzyme, and separating the reaction product by HPLC. Specifically, Taniguchi et al. (Taniichi, N., Nishikawa, A., Fujii, S., and Gu, J., Methods Enzymol. 179, 397-408 (1989); Nishikawa, A., Gu, J., Fujii, S., and Tanichichi, N. Biochim. Biophys. Acta 1035, 313-318 (1990)).

  That is, the enzyme activity of GnT-V can be measured using GnGn-2-aminopyridine (GnGn-biPA) as an acceptor and UDP-GlcNAc (Sigma) as a donor. The activity intensity is expressed as transferred N-acetylglucosamine / hour / mg protein. The amount of protein was determined using the BCA kit (Pierce Chemical Company, Rockford, Ill.) According to a previously reported report (Redinbaugh, M, G., and Turley, RB Anal. Biochem. 153, 267-271 (1986)). Albumin can be measured as a standard.

  Isolation and purification of the present enzyme from the culture supernatant can be performed by combining a plurality of methods commonly used for protein purification. In addition, as a useful purification method of the present enzyme, it is possible to utilize the affinity for an acceptor and a donor. In the present invention, the protein-free culture supernatant is first concentrated by the ultrafiltration method, and then the active fraction is obtained by hydrophobic chromatography using Phenyl-Sepharose.

  Further, after adsorption by Hydroxylapate, the active fraction was eluted and subjected to two affinity chromatography, an acceptor column chromatography (UDP-hexanolamine-Agarose) and a donor column chromatography (GnGn-Asn-Sepharose), and the reducing agent was removed. By performing sodium dodecyl sulfate-polyacrylamide electrophoresis in the presence, a polypeptide showing a single band can be obtained. Specific examples of these isolation and purification procedures are described in Example 2.

  As a result of the intensive study as described above, the present inventors concentrated and purified GnT by about 20,000-fold concentration from a concentrated protein-free culture supernatant of QG cells derived from human lung cancer (small cell carcinoma) by four steps of purification. -V was successfully isolated and purified, and the partial amino acid sequence of the enzyme was clarified. The enzyme according to the present invention is obtained for the first time by selecting a common protein isolation and purification method by a specific combination and performing it under specific conditions.

  Once purified and isolated, and its material and structural properties are elucidated as described below, these properties are used as indices, and any combination of conventional protein separation and purification methods is described in this specification. It can be purified by various methods different from the methods described below. Further, an appropriate synthetic probe is prepared based on the partial amino acid sequence of the enzyme disclosed by the present invention, and a bird, an amphibian, a mammal, etc., are isolated by a conventional method from a chromosome library or a cDNA library derived therefrom, and It is easy for those skilled in the art to produce isolated genes in large quantities by recombinant DNA technology.

The isolated and purified enzyme has the following enzymatic properties.
1. Action: Transfers N-acetylglucosamine from UDP-N-acetylglucosamine to α-6-D-mannoside.
2. Substrate specificity: Assuming that GnGn-bi-PA as a receptor is 100, reactivity of about 78% for GnGnF-bi-PA, about 125% for GnGnGn-tri-PA, and about 66% for GnM-PA Is shown.
3. Optimum pH; 6.2-6.3
4. Inhibition, activation and stabilization; activity does not require Mn ²⁺ . The activity is not inhibited even in the presence of 20 mM EDTA.

  5. Molecular weight; sodium dodecyl sulfate-polyacrylamide (SDS) electrophoresis analysis showed a single band having a molecular weight of about 73,000 under non-reducing conditions, and about 60,000 in addition to the above-mentioned bands under reducing conditions. A band having a small molecular weight is observed. In addition, when the isolated and purified enzyme is subjected to native polyacrylamide electrophoresis, and the band is cut out from the gel and the present enzyme activity is measured, the enzyme activity is recognized according to the concentration of the corresponding band. From the above, it was confirmed that the isolated and purified protein was the target GnT-V. A specific example is described in Example 3.

  In addition, each of the two bands in SDS-polyacrylamide electrophoresis under reducing conditions was cut out from the SDS-polyacrylamide electrophoresis gel, and after trypsin digestion, peptide mapping by reverse phase high performance liquid chromatography was attempted. Similar patterns were obtained for the patterns. From these results, it was concluded that both proteins may have undergone enzymatic cleavage, but were derived from the same protein. A specific example is described in Example 4.

6. Km value: The Km values for the acceptor GnGn-bi-PA and the donor UDP-GlcNAc are 133 μM and 3.5 mM, respectively.
7. Peptide fragment: The amino acid sequence was identified using the tryptic digested polypeptide fragment, and the following partial amino acid sequence was obtained.
(1) Thr-Pro-Trp-Gly-Lys
(2) Asn-Ile-Pro-Ser-Tyr-Val
(3) Val-Leu-Asp-Ser-Phe-Gly-Thr-Glu-Pro-Glu-Phe-Asn-His-Ala-Asn-Tyr-Ala
(4) Asp-Leu-Gln-Phe-Leu-Leu
(5) Asn-Thr-Asp-Phe-Phe-Ile-Gly

  Furthermore, a GnT-V according to the present invention is encoded by preparing an expected oligonucleotide using the above peptide fragment and screening a chromosome library or a cDNA library by a conventional method using the oligonucleotide as a probe. Genes can be obtained.

  The gene of the present invention may be any of cDNA, genomic DNA and chemically synthesized DNA. For the cDNA, for example, a DNA (nucleotide) primer designed based on the partial amino acid sequence shown in Example 5 of GnT-V purified from human cells, for example, QG cells derived from human lung cancer, as described above, is used. It can be cloned by the polymerase chain reaction (PCR). A specific example of cloning is shown in Example 8.

The gene of the present invention further includes DNA encoding a protein having GnT-V activity and hybridizing with the nucleotide sequence of SEQ ID NO: 8.
SEQ ID NO: 8 shows the nucleotide sequence of the DNA encoding GnT-V of the present invention cloned in Example 8, and the amino acid sequence deduced from the nucleotide sequence.

  Thus, once the amino acid sequence is determined, a polypeptide having one or more amino acids added to the natural amino acid sequence and still maintaining GnT-V activity, one or more amino acids from the natural amino acid sequence A polypeptide in which one or more amino acids in the natural amino acid sequence have been replaced by other amino acids and the GnT-V activity has been maintained. Various modified GnT-Vs, such as peptides, and polypeptides having a combination of the above-described amino acid addition modification, amino acid removal modification and amino acid substitution modification, and still maintaining GnT-V activity, are designed. , It can be manufactured.

  The number of amino acids in the modification such as addition, removal and substitution of the amino acid is not particularly limited. For example, the number of amino acids in the functional protein used in the hybrid protein with GnT-V of the present invention (for example, , Known proteins for extraction and purification or stabilization, such as maltose-binding protein, or various physiologically active proteins such as cytokines such as IL-3 and IL-11) and signals added to the present factor. It is determined depending on that of the peptide, ie, on the purpose of the modification. For example, addition of 1 to 30, preferably 1 to 10 can be mentioned.

  Regarding the removal, the number of amino acids to be removed is designed and determined so as to maintain the GnT-V activity, and is, for example, 1 to 30, preferably 1 to 20, and the number of amino acids other than the active region of the present factor. Include the number of amino acids in the region. Further, regarding the substitution, the number of amino acids to be substituted is designed and determined so as to maintain GnT-V, and includes, for example, 1 to 10, preferably 1 to 5.

  In the present invention, the nucleotide sequence shown in SEQ ID NO: 8 is disclosed as the nucleotide sequence of the gene encoding GnT-V, but the GnT-V gene of the present invention is not limited thereto. Once the amino acid sequence of native GnT-V has been determined or the amino acid sequence of a modified GnT-V has been designed, various nucleotide sequences encoding the same amino acid sequence are designed based on codon degeneracy, and Can be prepared. In this case, it is preferable to use codons that are frequently used depending on the host to be used.

  In order to obtain a gene encoding the natural GnT-V of the present invention, cDNA can be obtained as described in Example 8, but is not limited thereto. That is, once one nucleotide sequence encoding the amino acid sequence of natural GnT-V is determined, the gene encoding natural GnT-V can be cloned as cDNA using a strategy different from the strategy specifically disclosed in the present invention. And can also be cloned from the genome of the cell producing it.

  When cloning from the genome, various primer nucleotides or probe nucleotides used in Example 8 can be used as probes for selection of genomic DNA fragments. Further, another probe designed based on the nucleotide sequence described in SEQ ID NO: 8 can also be used. General methods for cloning the DNA of interest from the genome are well known in the art (Current Protocols In Molecular Biology, John Wiley & Sons, Chapters 5 and 6).

  The gene encoding the natural GnT-V of the present invention can also be prepared by chemical synthesis. Chemical synthesis of DNA is easy in the art by employing an automatic DNA synthesizer, such as Applied Biosystems' 396 DNA / RNA synthesizer. Therefore, those skilled in the art can easily synthesize the DNA having the nucleotide sequence shown in SEQ ID NO: 8.

  The gene encoding the natural GnT-V of the present invention with a codon different from the native codon and the gene encoding the modified GnT-V can also be prepared by chemical synthesis as described above, and SEQ ID NO: 8. Can be obtained according to a conventional method such as site-directed mutagenesis using DNA or RNA having the nucleotide sequence shown in Table 1 as a template together with a mutagenic primer (for example, Current Protocols In Molecular Biology, John Wiley & Sons). , See Chapter 8).

  When the GnT-V gene of the present invention is obtained as described above, it can be used to produce recombinant GnT-V by a conventional gene recombination method. That is, DNA encoding the GnT-V of the present invention is inserted into an appropriate expression vector, the expression vector is introduced into an appropriate host cell, the host cell is cultured, and the resulting culture (cell or medium) )) To collect the desired GnT-V.

Prokaryotes or eukaryotes can be used as hosts. As prokaryotes, bacteria, particularly Escherichia coli , Bacillus bacteria, such as B. subtilis, can be used. Yeast As eukaryote, such as Saccharomyces (Saccharomyces) genus yeasts, e.g. Saccharomyces cerevisiae (S. Serevisiae), eukaryotic microbes, insect cells etc., for example, Yoga cells (Spodoptera frugiperda), cabbage looper cells (Trichoplusia ni ), Silkworm cells ( Bombyx mori ), and animal cells such as human cells, monkey cells, mouse cells, and the like. In the present invention, an organism itself, for example, an insect such as a silkworm or a cabbage looper can also be used.

  As the expression vector, a plasmid, a phage, a phagemid, a virus (baculo (insect), vaccinia (animal cell)) and the like can be used. The promoter in the expression vector is selected depending on the host bacterium. For example, a lac promoter, a trp promoter or the like is used as a bacterial promoter, and an adhl promoter or a pqk promoter is used as a yeast promoter. Insect promoters include the baculovirus polyhedrin promoter, and animal cells include Simian Virus 40 early or late promoters.

Transformation of a host with an expression vector can be performed by conventional methods well known in the art, and these methods are described, for example, in Current Protocols in Molecular Biology, John Wiley & Sons. Culture of the transformant can also be performed according to a conventional method.
Purification of GnT-V from the culture can be carried out according to a conventional method for isolating and purifying a protein, for example, by ultrafiltration, various column chromatography, for example, chromatography using Sepharose, or the like.

Hereinafter, the present invention will be described in detail with reference to examples.
Embodiment 1 FIG . Cultivation of GnT-V Producing Cells and Preparation of Culture Supernatant QG cells derived from human lung cancer (small cell carcinoma) (Microtechnological Research Institute, Inc. No. 3967) are cultured in Opticell 5300 (Charles River Inc., Wilmington, Mass.). did. ² × 10 ⁹ cells are inoculated in a porous ceramic growth chamber (effective surface area> 32,000 cm ² ) and cultured using Ham, sF-12 culture medium (Flow) containing 5% calf serum (GIBCO) until confluence. did.

Next, the serum amount was gradually reduced by continuously adding a Ham, s F-12 culture solution containing no protein containing 10 ^-8 M sodium selenite. After 2 weeks of culture, 140 L of culture supernatant was obtained. This culture supernatant was concentrated about 100-fold using an ultrafiltration apparatus using a UF-membrane (Milipore, Bedford, MA) as a filtration membrane. 1.4 liters of the concentrated culture supernatant was used as a purified material.

Embodiment 2 FIG . Purification of this enzyme from concentrated culture supernatant (1) Preparation of UDP-hexanolamine agarose and GlcNAcβ1-2Manα1-3- (GlcNAcβ1-2Manα1-6) Manβ1-4GlcNAcβ1-4GlcNAc-asparagine (GnGn-Asn) sepharose -Hexanolamine agarose was purchased from Sigma (St. Louis, MO). GnGn-Asn was prepared from human transferrin. First, human transferrin was repeatedly digested twice with pronase (Boehringer Mannheim) for 12 hours at 37 ° C. in 0.1 M borate buffer (pH 8.0), and then equilibrated with 10 mM acetate buffer (pH 6.0) in advance. The glycopeptide was separated by a gel filtration column (Toyoperl HW-40, 2.6 × 100 cm, Tosoh, Tokyo).

Next, GnGn-Asn was obtained by enzymatically digesting the separated glycopeptide with sialitases ( Arthrobacter ureafacience , Nacalai Tesque, Kyoto) and β-galactosidase (jackbean, Seikagaku Co, Japan). GnGn-Asn Sepharose was obtained by binding 100 μmol of GnGn-Asn by reacting 10 ml of activated CH-Sepharose (Pharmacia).

(2) Phenyl-Sepharose column chromatography
A 4.5 × 18 cm column was packed with 280 ml of phenyl-Sepharose and equilibrated with 1.5 L of 20 mM potassium phosphate buffer (pH 6.8, containing 40% saturated ammonium sulfate). 1.3 liters of the concentrated cell culture supernatant prepared in Example 1 was made 40% saturated with ammonium sulfate, and after centrifugation at 3000 g for 30 minutes, the supernatant was adsorbed to the column.

  Elution was performed using a 20 mM potassium phosphate buffer solution (pH 6.8) containing 40% ammonium sulfate in a linear gradient in which the ammonium sulfate concentration was reduced to 0%. The flow rate at that time was 3 ml / min. The absorbance at 280 nm and the GnT-V enzyme activity of the eluted fraction were measured. As shown in FIG. 1, the peak of the GnT-V enzyme activity was eluted later than the absorption peak at 280 nm.

(3) Hydroxyapatite column chromatography GnT-V enzymatic activity fraction eluted from phenyl-sepharose column chromatography was collected, further concentrated using Amicon YM-30 membrane, and simultaneously with 20 mM potassium phosphate buffer (pH 6.0). The solvent exchange with 8) was performed. This was adsorbed on 55 ml of hydroxyapatite which had been equilibrated with 300 ml of 20 mM potassium phosphate buffer (pH 6.8). Elution was performed with a linear gradient of 50 mM-300 mM potassium phosphate buffer (pH 6.8) at 3 ml / min. FIG. 2 shows the elution pattern of hydroxyapatite chromatography. The GnT-V enzyme activity peak was separated and eluted from most other proteins.

(4) UDP-Hexanolamine agarose-column chromatography Next, the active fractions from the hydroxyapatite column were collected, and the solvent was converted to 10 mM potassium phosphate buffer (pH 6.25) using Amicon YM-30 membrane. This was adsorbed on a 20 ml UDP-hexanolamine agarose column which had been pre-equilibrated with 100 ml of 10 mM potassium phosphate buffer (pH 6.25). The enzyme was eluted by flowing a 10 mM potassium phosphate buffer (pH 6.25) containing 0.3 M NaCl at a flow rate of 15 ml / hour. This elution pattern is shown in FIG.

(5) GnGn-Asn Sepharose-column chromatography Finally, GnGn-Asn Sepharose-column chromatography was performed. The active fraction from the UDP-hexanolamine agarose-column was adsorbed on 4 ml of GnGn-Asn Sepharose equilibrated with 20 ml of 10 mM potassium phosphate buffer (pH 6.25) containing 0.3 M NaCl and washed with the same buffer. Thereafter, elution was performed at a flow rate of 3 ml / hour of 30 mM Tris-HCl (pH 9.0) containing 0.3 M NaCl. The result is shown in FIG.
Table 1 shows the purification yields thus far. Each step was finally purified by about 20,000-fold with a yield of 70% or more.

Embodiment 3 FIG . SDS-polyacrylamide electrophoresis and native polyacrylamide electrophoresis SDS-polyacrylamide electrophoresis, the method previously reported (Laemmli, U.K., Nature, 227,680-685 , (1970)) in accordance with, 10-15% of the gradient The reaction was performed using polyacrylamide gel under reducing and non-reducing conditions. α-Lactoalbumin, Soybean trypsin inhibitor, Carbonic anhydrase, Ovalbumin, Bovine serum albumin, Phosphorylase b as molecular weight 14,400, 20,100,30,000,44,000 for markers, molecular weight 14,400,20,000,43,000, respectively. Was.

FIG. 5 shows a photograph of this gel in which the final purified enzyme was stained with Coomassie brilliant blue. SDS polyacrylamide electrophoresis under non-reducing conditions confirmed a single band of 73 kDa based on the molecular weight markers described above. On the other hand, SDS polyacrylamide electrophoresis under reducing conditions confirmed a band of 60 kDa other than 73 kDa on the gel. FIG. 6 shows a photograph of a gel stained with Coomassie brilliant blue in native polyacrylamide electrophoresis and the present enzyme activity cut out from the native polyacrylamide electrophoresis gel. This enzyme activity was observed according to the band intensity.
From the above, it was confirmed that the isolated and purified protein was the target GnT-V.

Embodiment 4 FIG . Peptide mapping analysis SDS polyacrylamide electrophoresis was performed under reducing conditions, and bands corresponding to 60 kDa and 73 kDa were cut out from the gel, respectively. A 30 mM Tris-HCl buffer (pH 6.5) containing 0.5% SDS was added to the sliced gel pieces, and after boiling, proteins were extracted with a handmade electric extraction device. After the extraction, 200 ng of trypsin (Sigma, St Louis, MO) dissolved in 50 mM Tris-HCl buffer (pH 8.0) was reacted at 37 ° C. for 11 hours.

  After enzyme digestion, the mixture is applied to a reversed-phase high-performance liquid chromatography column (Chemcosorb 3 ODS-H, Osaka, Japan, 2.1 × 75 mm), and eluted with a gradient of 0-60% acetonitrile under acidic conditions (0.1% TFA). did. The elution pattern at that time is shown in FIG. The 60 kDa and 73 kDa elution patterns showed similar patterns, concluding that both proteins may have undergone some enzymatic digestion, but consisted mostly of a common polypeptide.

Embodiment 5 FIG . Determination of Partial Amino Acid Sequence In order to know the amino acid sequence, the purified GnT-V peptide fragment obtained in Example 2 was subjected to a Gas-phase sequencer (Applied Biosystem Inc., Foster City, Calif.). A peptide fragment was obtained.
(1) Thr-Pro-Trp-Gly-Lys
(2) Asn-Ile-Pro-Ser-Tyr-Val
(3) Val-Leu-Asp-Ser-Phe-Gly-Thr-Glu-Pro-Glu-Phe-Asn-His-Ala-Asn-Tyr-Ala
(4) Asp-Leu-Gln-Phe-Leu-Leu
(5) Asn-Thr-Asp-Phe-Phe-Ile-Gly

Embodiment 6 FIG . Substrate specificity Using the following sugar acceptors, the substrate specificity was measured in accordance with the aforementioned method for measuring enzyme activity.

  GnGnF-, GnGF-, GGnF-, Gn (Gn) GnF-, Gn (Gn) Gn-, GG-, GnGn-, MM- and GnM- were obtained by treating bovine IgG with anhydrous hydrazine, followed by the method of Hase et al. Hase, s., Ibuki, T., and Ikehara, T., J. Biochem. 95, 197-203 (1984)). The purified GnT-V was diluted with PBS buffer to a final concentration of 0.1 mg protein / ml, and then reacted with each fluorescently labeled sugar receptor. The results are shown in Table 2.

  This enzyme was found to react with any of GnGnF-bi-PA, GnM-PA and GnGnGn-tri-PA.

Embodiment 7 FIG . Optimum pH
Assays containing 0.2 M GlcNAc, 40 mM EDTA, 1% Triton X-100, 40 mM UDP-GlcNAc in 0.25 M potassium phosphate buffer at pH 6.0, 6.25, 6.5 and 7.0. A mixture was prepared.
15 μl of a test sample and 10 μl of a substrate (GnGn-bi-PA) were added to 25 μl of the assay mixture, and the mixture was incubated at 37 ° C. for 4 hours. FIG. 8 shows the results.

Embodiment 8 FIG . Isolation and identification of GnT-V cDNA and preparation of expression vector Based on the amino acid sequence represented by SEQ ID NO: 3 obtained in Example 5, oligomer S18 represented by Sequence Listing and SEQ ID NO: 6 was synthesized and further distributed. Based on the amino acid sequence represented by Sequence Listing and SEQ ID NO: 5, oligomer A21 represented by Sequence Listing and SEQ ID NO: 7 was synthesized.

  Double-stranded cDNA was synthesized from RNA having poly A of QG cells using a cDNA Synthesis kit (Pharmacia). Next, PCR was performed using Ampli-Taq (manufactured by Takara) using this cDNA as a template and oligomers S18 and A21. As a result, a DNA fragment of about 500 base pairs, which is a GnT-V cDNA fragment, was obtained.

Next, using the obtained cDNA fragment as a probe, the cDNA was further cloned. That is, using this cDNA as a template, α ³² P-CTP labeling was performed using a random primer labeling kit (Amersham). Using the obtained probe, a human fetal liver cDNA library (Clontech) was screened by a conventional method. Among the obtained clones, the longest clone was excised with EcoRI and subcloned into pBluescript II ks (+) (Strategene). Due to the EcoRI site in the insert, two fragments were obtained. As a result of sequencing each of the two fragments, a B fragment of about 800 bp and an F fragment of about 1200 bp were obtained.

The sequence of the DNA fragment obtained as described above was determined by Sequenase (manufactured by USB), and the result is shown in SEQ ID NO: 8 in Sequence Listing. It was confirmed that this DNA fragment contained all the nucleotide sequences encoding the five amino acid sequences obtained in Example 5.
The GnT-V cDNA fragment obtained this time does not reach the termination codon at the 3 'end. Therefore, although it is clear that there is an undetermined base pair at the 3 ′ end, it is easy for those skilled in the art to determine the undetermined base pair.

Next, the GnT-V cDNA fragment was incorporated into an expression vector for expression in animal cells. That is, after the expression vector pSVK3 (Pharmacia) was digested enzymatically with EcoRI and SmaI, the F fragment was cut out at the EcoRI site and the SmaI site in the insert and inserted (see FIG. 9).
Next, the vector into which the F fragment was inserted was digested with EcoRI, treated with CIP (Calf intestinal phosphatase) to prevent self-ligation, and inserted into the expression vector by inserting the B fragment cut out at the EcoRI site.

Embodiment 9 FIG . Expression of Cloned GnT-V cDNA and Enzyme Activity of Expressed Protein Using the expression vector prepared in Example 8, a GnT-V cDNA fragment was expressed in animal cells, and whether the expressed protein had GnT-V enzyme activity or not. Was considered.
COS-1 cells were used as expression animal cells, and an expression vector containing a GnT-V cDNA fragment was transfected by electroporation. That is, about 5 × 10 ⁶ COS-1 cells prepared in advance were suspended in HEBS buffer (20 mM Hepes, 137 mM NaCl, 5 mM KCl, 0.7 mM Na ₂ HPO ₄ , 6 mM Dexrose, pH 7.05), and The plasmid (60 μg) was added, and the final volume was 800 μl, which was performed using an electroporator (manufactured by Bio-Rad) at 250 V and 960 mF.

Next, the entire amount of COS-1 cells transfected with the GnT-V gene was spread on a petri dish having a diameter of 6 cm, and cultured at 37 ° C. in the presence of 5% CO ₂ for 48 hours. The cultured cells were collected with a rubber policeman, suspended in 50 μl of PBS buffer, and then disrupted with a sonicator. Thereafter, the supernatant was collected by centrifugation, and the enzyme activity of 15 μl of the supernatant was measured by the method described above. FIG. 10 shows the measurement results.
From the above results, it was confirmed that the GnT-V cDNA fragment cloned this time contains the minimum length required for active expression.

Effect of the Invention UDP-N-acetylglucosamine: α-6-D-mannoside, β1,6-N-acetylglucosaminyltransferase was isolated from a protein-free culture supernatant of QG cells derived from human lung cancer (small cell carcinoma). After purification, its properties and partial amino acid structure were determined. In addition, the present inventors have clarified the substrate specificity for various sugar receptors using the enzyme purified for the first time this time. Glycosyltransferases having these specificities are one of the important enzymes responsible for regulating the sugar chain biosynthetic pathway in vivo. In addition, it is becoming clear that the present enzyme is involved in sugar chain modification as cells become cancerous or malignant.

  Therefore, the enzyme according to the present invention not only has diagnostic utility as a marker of malignancy of cancerous cells, but also establishes a screening system for a specific inhibitor of the enzyme using the enzyme. This makes it possible for the first time to design a cancer metastasis inhibitor. Further, since the β (1,6) branch structure can be introduced into various sugar receptors exemplified using the present enzyme, oligosaccharides having a β (1,6) branch structure as a reaction product can be industrially produced. Can now be manufactured. In addition, from the viewpoint of sugar chain engineering, by producing the present enzyme in large quantities, it is possible to uniformly produce sugar chains in the production of useful substances by genetic manipulation.

It is a graph which shows the elution pattern of a phenyl sepharose column chromatography. The solid circle shows the protein elution pattern, and the solid circle shows GnT-V activity. Elution was performed by reducing the concentration of ammonium sulfate from 40% to 0%. The ammonium sulfate concentration is indicated by a dotted line. It is a graph which shows the elution pattern of a hydroxyapatite column chromatography. The solid circle shows the protein elution pattern, and the solid circle shows GnT-V activity. The arrow in the figure indicates the starting point of the elution buffer. Elution was performed by forming a concentration gradient of 50 mM to 300 mM phosphate buffer. The concentration of the phosphate buffer is indicated by a dotted line. It is a graph which shows the elution pattern of UDP-hexanolamine agarose-column chromatography. The solid circle shows the protein elution pattern, and the solid circle shows GnT-V activity. The arrow in the figure indicates the starting point of the elution buffer. Elution was performed by flowing 0.3 M NaCl at 3 ml / hour. It is a graph which shows the elution pattern of GnGn-Asn Sepharose-column chromatography. The solid circle shows the protein elution pattern, and the solid circle shows GnT-V activity. The arrow in the figure indicates the starting point of the elution buffer. Elution was performed by flowing 0.3 M NaCl at 3 ml / hour. It is a photograph instead of a drawing showing the result of SDS polyacrylamide electrophoresis. It is a photograph instead of a drawing showing the result of native polyacrylamide electrophoresis. It is a figure which shows the result of a peptide mapping analysis. 3 is a graph showing the optimum pH of the enzyme of the present invention. FIG. 9 is a diagram showing the construction of the expression vector of the present invention. FIG. 10 is a high-performance liquid chromatography elution diagram showing that a product (GnGnGn-tri'-PA) was produced from a substrate (GnGn-bi-PA) by GnT-V, which is an expression product of the present invention. .

Claims (6)

  A β1,6-N-acetylglucosaminyltransferase having an amino acid sequence comprising at least the amino acid sequence represented by SEQ ID NO: 8, or an amino acid sequence in which one or more amino acids are modified in this amino acid sequence.   A DNA encoding the β1,6-N-acetylglucosaminyltransferase according to claim 1.   The DNA according to claim 3, which has the nucleotide sequence shown in SEQ ID NO: 8.   An expression vector comprising the DNA according to claim 2.   A host transformed with the expression vector according to claim 4.   The method for producing a β1,6-N-acetylglucosaminyltransferase enzyme according to claim 1, wherein a host transformed with an expression vector containing a DNA encoding the enzyme is cultured or bred, and the host is transformed from the host. A method comprising collecting the enzyme.