JP2839232B2 - New sugar chain synthase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sugar chain synthase and a DNA encoding the enzyme. More specifically, the present invention relates to N-acetylgalactosamine α 2,6 sialyltransferase (GalNAcα2,6-sialyltransferase) and DNA encoding the enzyme. The enzyme is useful as a drug having an inhibitory effect on cancer metastasis and virus infection, or as a reagent for increasing a physiological action by adding sialic acid to the drug.

[0002]

2. Description of the Related Art Sialic acid is used, for example, in cell-cell communication,
It is a substance that controls important physiological actions such as cell-substrate interaction and cell adhesion. It is known that there are a variety of different cell surface sialic acids that are regulated during processes such as development, differentiation, and oncogene transformation. Sialic acid is located at the terminal position of the carbohydrate group of glycoproteins and glycolipids.
Enzymatically introduced from CMP-Sia into these sites. For example, glycoproteins have three binding modes: Siaα2,6Ga
l, Siaα2,3Gal and Siaα2,6GalNAc are commonly present (Hakomori, S., Ann. Rev. Biochem., 50, 7
33-764, 1981), gangliosides frequently have two binding modes, Siaα2,3Gal and Siaα2,8Sia (Fishman, P., and Brady, RO, Science,
194, 906-915, 1976).

[0003] The enzyme responsible for the enzymatic introduction of sialic acid (sialyltransferase) is sialyltransferase (sialyltransferase).
ferases). It is known that at least 12 different sialyltransferases are required to synthesize all previously known sialyl oligosaccharide structures (Broquet, P. et al., In
t. J. Biochem., 23, 385-389, 1991; and Weinstei
n, J. et al., J. Biol. Chem., 262, 17735-17743, 19
87). Of these, five types of sialyltransferases have been purified, and all enzymes are known to exhibit high specificity for each receptor substrate (Sadler, J. et al.,
J. Bio.Chem., 254, 4434-4443, 1979; Weinstein, J.
et al., J. Biol. Chem., 257, 13835-13844, 1982; R
earick, J. et al., J. Biol. Chem., 254, 4444-4451,
1979; and Joqiasse, DH eal., J. Biol. Chem.,
260, 4941-4951, 1985).

[0004] cDNA encoding the above sialyltransferase
About Galβ1,4GlcNAc α2,6-sialyltransferase
CDNA encoding (Galβ4GlcNAc-α6ST) has been cloned from various tissues including liver (Weinste
in, J. et al., J. Biol. Chem., 262, 17735-17743, 1
987; Grundmann U. et al., Nucleic Acids Res. 18,66
7, 1990; Bast, B. et al., J. Cell. Biol., 116, 423
-435, 1992; and Hamamoto, T. et al., Bioorg. An
d Medic. Chem., 1, 141-145, 1993). Gal β1,3
GalNAc α2,3-sialyltransferase (Galβ3GalNAc-α3ST)
(Gillespie, W. et al., J. Biol. Ch.
em., 267, 21004-21010, 1992; and Lee, Y. et a
l., Eur. J. Biochem, 216, 377-385, 1993), Gal β
1,3 (4) GlcNAcα2,3-sialyltransferase (Galβ3 (4) GlcNAc
-cDNA encoding α3ST) (Wen, DX et al., J. Biol.
Chem., 267, 21011-21019, 1992) has also been cloned.

[0005] However, cDNA encoding GalNAcα2,6-sialyltransferase (EC 2.4.99.3; GalNAc-α6ST) has not been cloned. There is also a report that GalNAcα2,6-sialyltransferase was isolated (Hakomori, S., Ann. Rev.
Biochem., 50, 733-764, 1981), but it was not purified to the extent that it could be identified as a substance, and had problems in reaction specificity, stability of properties, and supply, and could not be put to practical use.

Accordingly, an object of the present invention is to provide a purified GalNAcα2,6-sialyltransferase. The present invention also relates to GalNAcα2,6-
Cloning cDNA encoding sialyltransferase,
An object of the present invention is to provide a DNA sequence encoding the GalNAcα2,6-sialyltransferase and an amino acid sequence of the enzyme. Another object of the present invention is to express a portion of the structure of the above GalNAcα2,6-sialyltransferase involved in activity as a protein in a large amount.

[0006]

Means for Solving the Problems The present inventors have made intensive efforts to achieve the above-mentioned object, and obtained GalNAcα2,6-
Cloning cDNA encoding sialyltransferase,
The present invention has been completed. That is, the present invention is specified by the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
GalNAcα2,6-sialyltransferase P-B1. Further, the present invention relates to a GalNAcα2,6-sialyltransferase gene encoding the amino acid sequence of the above GalNAcα2,6-sialyltransferase, and a nucleic acid No. 1 represented by SEQ ID NO: 1 in the sequence listing which is one embodiment thereof. The present invention provides a GalNAcα2,6-sialyltransferase gene having the nucleotide sequence specified by Nos. To 1698. Furthermore, a recombinant vector containing the above GalNAcα2,6-sialyltransferase gene, a plasmid λ CEB-3034 which is one embodiment thereof, a transformant transformed with the recombinant vector, and SEQ ID NO: 1 in the sequence listing Also provided is a GalNAcα2,6-sialyltransferase active domain specified by 233-566 of the amino acid sequence shown in.

Further, according to the present invention, the above GalNAcα2,6-
An extracellular secretory protein containing a polypeptide portion, which is an active domain of sialyltransferase, and a signal peptide, which catalyzes GalNAcα2,6-sialyltransferase, and as an embodiment thereof, SEQ ID NO: 2 in the sequence listing. Provided is a protein SB-690 specified by the amino acid sequence shown in SEQ.
Further, a gene encoding the above protein, and as one embodiment thereof, nucleic acids 1 to 10 shown in SEQ ID NO: 2 in the sequence listing.
A gene having the nucleotide sequence represented by 65, a recombinant vector containing the gene, and a plasmid pcDSB-690 as one embodiment thereof are provided. Further, the transformant transformed by the recombinant vector, and culturing the transformant, characterized in that the protein is collected from the culture, catalyzes the GalNAcα2,6-sialyl transfer. A method for producing a protein is also provided.

The most preferred example of the GalNAcα2,6-sialyltransferase of the present invention is GalNAcα2,6-sialyltransferase P
-B1 is provided. Hereinafter, GalNAcα2,6-sialyltransferase P-B1 will be described in detail as an example of the enzyme of the present invention, but the GalNAcα2,6-sialyltransferase of the present invention is GalNAcα2,6-sialic acid. Not limited to the transferase P-B1, GalNAcα2, the active domain of the 6-sialyltransferase P-B1 revealed for the first time according to the present invention, or a modified or modified GalNAcα2, part of the amino acid sequence of the amino acid sequence thereof. It is to be understood that all GalNAcα2,6-sialyltransferases having a 6-sialyltransferase active domain are included within the scope of the present invention. Preferred examples of such an active domain include SEQ ID NO: 1 in the sequence listing.
Ga specified by 233-566 of the amino acid sequence disclosed in
Examples include the lNAcα2,6-sialyltransferase active domain.

For example, GalNAcα2,6-sialyltransferase P
Methods for isolating the cDNA encoding -B1 are described in detail in the Examples below. However, the method for isolating the cDNA encoding GalNAcα2,6-sialyltransferase P-B1 is not limited to these methods, and those skilled in the art can refer to the methods described in the following Examples, By appropriately modifying or changing this method, the desired cDNA can be easily isolated. It encodes GalNAcα2,6-sialyltransferase P-B1 described in SEQ ID NO: 1 in the following sequence listing.
Although DNA is a preferred embodiment of the present invention, the GalN
The DNA encoding Acα2,6-sialyltransferase P-B1 is not limited to this specific embodiment, and the amino acid sequence of GalNAcα2,6-sialyltransferase P-B1 disclosed by the present invention is All encoding DNAs are encompassed by the present invention.

Furthermore, GalNAcα2,6-sialyltransferase P-
DNAs encoding GalNAcα2,6-sialyltransferase active domains in which the active domain of B1 or a part of the amino acid sequence thereof has been modified or modified are also included in the scope of the present invention. For example, a DNA encoding the GalNAcα2,6-sialyltransferase active domain identified by 233-566 of the amino acid sequence disclosed in SEQ ID NO: 1 of the Sequence Listing is a preferred embodiment of the present invention. The DNA specified by 699-1698 of the nucleic acid sequence disclosed in SEQ ID NO: 1 in the sequence listing is a particularly preferred embodiment of the present invention.

The GalNAcα2,6-sialyltransferase P of the present invention
-B1 may remain in cells after expression and may not be secreted extracellularly. Also, when the intracellular concentration exceeds a certain level,
It is possible that the expression level of the enzyme is reduced. Ga above
In order to effectively utilize the GalNAcα2,6-sialyltransferase activity of lNAcα2,6-sialyltransferase P-B1, a soluble form of the protein that is secreted from cells during expression must be maintained while maintaining the activity of the enzyme. Can be manufactured. Examples of such a protein include an extracellular secretory type containing a signal peptide and a polypeptide portion which is a GalNAcα2,6-sialyltransferase active domain involved in the activity of the above GalNAcα2,6-sialyltransferase P-B1. Proteins that catalyze the transfer of GalNAcα2,6-sialyl acid can be mentioned.

The sialyltransferase cloned so far has the same domain structure as other glycosyl-transferases. That is, a short cytoplasmic middle tail at the NH ₂ terminus, a hydrophobic signal anchor domain,
Proteolytically sensitive stem region, and COOH-
It has a large active domain at the end (Paulson, JC and
Colley, KJ, J. Biol. Chem., 264, 17615-17618, 1
989). In order to determine the location of the transmembrane domain of the GalNAcα2,6-sialyltransferase P-B1 of the present invention, kites and Doolittle (Kyte, J. and Doolittle, RF, J. Mol. Bi)
, 157, 105-132, 1982) can be used. For estimating the active domain portion, a recombinant plasmid into which various fragments have been introduced can be prepared and used. An example of such a method is described in detail in the examples of the present specification, but the method of confirming the location of the transmembrane domain and estimating the active domain portion is not limited to this method.

In order to produce an extracellular secretory protein containing a polypeptide portion which is the active domain of GalNAcα2,6-sialyltransferase P-B1 and a signal peptide, for example, an immunoglobulin signal peptide sequence is used as a signal peptide. The sequence corresponding to the active domain of GalNAcα2,6-sialyltransferase P-B1 may be fused in-frame to the signal peptide. As such a method, for example, the method of Jobrin (Jobling, SA and Geh
rke, L., Nature (Lond.), 325, 622-625, 1987), and the specific method is described in detail in the examples of the present specification. However, the type of signal peptide and the method of binding the signal peptide to the active domain are not limited to the above methods, and those skilled in the art
The polypeptide portion that is the active domain of α2,6-sialyltransferase P-B1 can be appropriately selected, and extracellular secretory type is obtained by binding them to any available signal peptide by an appropriate method. Protein can be produced.

Hereinafter, the present invention will be described specifically with reference to Examples, but the scope of the present invention is not limited to these Examples.

[Example] cDNA of GalNAcα2,6-sialyltransferase P-B1
To obtain clones, PCR with two degenerate oligonucleotides (ST-107 and ST-205) was performed using chicken embryo cDNA as template. A fragment of the desired size of about 150 bp was obtained. Among the PCR recombination products, clones called CEB1 were Gal β4GlcNAc-α6STRL (180-225 residues), Gal β3 (4) GlcNAc-α3STRL (158-203 residues) and
Gal β3GalNAc-α3STPS (144-189 residues) had a unique amino acid sequence different from the known sialyl motif. Gal β4GlcNAc-α6S, a sialyl motif of CEB1
TRL, Gal β3 (4) GlcNAc- α3STRL and Gal β3GalNAc-
The homology between α3STPS and CEB1 was 56%, 58% and 6%, respectively.
0%.

6 using cDNA insert from CEB1
A day-old chicken embryo cDNA library was screened and several cDNA clones were identified. Among them, clone λCEB-3043 contained a 2.7 kb insert (Figure 1). In order to obtain another overlapping clone, random primed cDNA library was added to λCEB-30.
Rescreening was performed by hybridization to 43 5'-terminal 0.8 kb EcoRI-BglII fragments. Fifteen clones were isolated from this cDNA library. Clone λCEBHAD contained a 220 bp insert overlapping 160 bp with the 5'-end of clone λCEB-3043.

The combination of these two cDNAs is
1.7 kb ending with TGA stop codon at nucleotide 1699
It included an open reading frame. A polyadenylation signal (AATAAA), 23 nucleotides upstream from poly (A), is located at the 3 'end. Translation of this open reading frame results in a methionine residue at nucleotide 1 (K
ozak, M., Nature (Lond.), 308, 241-246, 1984) and a GalNAcα2,6-sialyltransferase P-B1 (P-B1) of the present invention, which is a protein consisting of 566 amino acids with a molecular weight of 64,781. B1) is expressed. CDNA containing a gene encoding GalNAcα2,6-sialyltransferase of the present invention, GalNAc of the present invention
λCE, a gene encoding α2,6-sialyltransferase
The nucleotide sequence of B-3043 and the amino acid sequence of GalNAcα2,6-sialyltransferase P-B1 of the present invention are shown in SEQ ID NO: 1 in the sequence listing.
Shown in

Polymerase chain reaction (PCR) PCR is performed using Gal β4GlcNAc-α6STRL (Weinstein, J. et al.,
J. Biol. Chem., 262,17735-17743, 1987), Gal β4Gl
cNAc-α6STHP (Grundmann, U. et al., Nucleic Acids R
es., 18, 667, 1990), and Gal β3GalNAc-α3STPS (Gil
lespie, W. etal., J. Biol. Chem., 267, 21004-2101
0, 1992) degenerate primer [5 ′
Primer ST107: TGGGCCTTGGII (A / C) AGGTGTGCTGTTG and 3 ′ primer ST205: AGGCGAATGGTAGTTTTTG (A / T) GC
CCACATC]. To obtain cDNA, poly (A) -rich RNA (2 μg) from 3-day-old chick embryos was
-dT primer (Pharmacia); dATP, dCTP, dGTP
And dTTP together with 1 mM; and 2 U / μl RNase inhibitor (Promega) together with 50 μl of 10 mM Tris-HCl
(pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ and 0.001% gelatin.
U Moloney murine leukemia virus reverse transcriptase (BRL)
And further incubated at 42 ° C. for 60 minutes.

After heating the above reaction solution at 94 ° C. for 3 minutes,
CDNA prepared from 0.2 μg poly (A) -rich RNA was
10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.25 m in μl
M MgCl ₂ , 0.001% gelatin, dATP, dCTP, dGTP and dT
TP was used for PCR experiments in a mixture containing 200 μM each, Taq DNA polymerase (Promega) 2 U, and 40 pmole of each PCR primer. 35 cycles
PCR amplification was performed, each cycle consisting of heat denaturation at 96 ° C. for 45 seconds; annealing at 50 ° C. for 60 seconds; and extension at 72 ° C. for 60 seconds. The PCR product was run on a 3% agarose gel and the DNA fragment corresponding to 150 bp was eluted from the gel (QIAX Kit, Qiagen), blunt-ended, kinased, and subcloned into the SmaI site of pUC119. And finally sequenced.

Construction of cDNA Library Total RNA was prepared from chick embryos (6 days old) by the guanidinium thiocyanate method: followed by centrifugation in 5.7 M CsCl solution (Sambrook, J., Molecular C
loning: a Laboratory Mannual, 2nd edition, Cold Sp
ring Harbor Laboratory, Cold Spring Harbor, NY 1
989). Poly (A) -rich RNA is purified by Oligotex-dT30 (Takara Shuzo) and used to create a cDNA library using λZAPII (Stratagene) and cDNA synthesis kit (Pharmacia) as oligo-dT primers and random primers. did.

Screening of cDNA Library The amplified cDNA library (1 × 10 ⁶ plaques) was screened with the chicken embryo PCR fragment. The filters were transferred plaques, 5 × SSC, 0.2% SDS , 5 × Denharudo solution and 10 [mu] g / ml denatured salmon sperm DNA ³² P
-Hybridized with radiolabeled DNA probe at 65 ° C for 12 hours. Thereafter, the plate was washed twice in 2 × SSC, 0.1% SDS at 65 ° C. for 20 minutes. To obtain plasmids from the isolated phage clones, phage midless rescue was performed according to the manufacturer's instructions for the λZAPII cloning kit (Stratagene). The cDNA insert was cut directly as a Bluescript plasmid. The construction of the plasmid was performed according to Sambrook, J. et al.,
Molecular Cloning: a Laboratory Manual, 2nd editi
on, Cold Spring Harbor Laboratory, Cold spring Har
bor, NY, 1989).

DNA Sequence Analysis The DNA sequence of the above insert was converted to a dideoxy chain termination method (Sang
er, F. et al., Proc. Natl. Acad. Sci. USA, 74, 546.
3-5467, 1977). Sequencing reaction and electrophoresis were performed using an auto-read DNA sequencing kit and a DNA sequencer (Pharmacia). Single-stranded DNA was prepared from E. coli XL-Blue (Stratagene) after superinfection with helper phage R408 (Stratagene). Sequence data was analyzed with a computer using PC / Gene (Teijin System Technology).

To confirm the presence of Northern and Southern blotting genes, Southern blot analysis of chicken genomic DNA was performed. EcoRI c of λCEB-201
Perform hybridization with the DNA insert,
A single band, Hind, for DNA cut with coRI and BamHI.
Two bands were obtained with DNA digested with III and SacI. From this simple hybridization pattern, it is clear that the cloned cDNA is a single copy gene. The details of the experiment are as follows.

Further, Northern blot hybridization was performed to examine the transcription pattern during the embryonic development process. 6, 8, and 10-day-old chick embryo-derived RN
Analysis of A revealed that two RNs of 3.0 and 2.2 Kb
A was detected. The 3.0 Kb transcript was predominant and was always expressed during the entire embryonic development. Low levels of 2.2 Kb transcript were detected in 6-day-old embryos, but were absent from 8 and 10-day-old embryos. Analysis of gene expression using 10 μg of poly A-rich RNA from chicken brain, heart, liver, embryo, kidney, and testis showed very low levels.
Transcripts of 3.0 and 4.0 Kb were detected in testis, but hardly detected in other tissues. The details of each experiment are as follows.

For Northern blotting, 5 μg of denatured poly (A) -rich RNA from chick embryos was subjected to size fractionation on formaldehyde-agarose gel, followed by Hybond N +
It was blotted on a nylon membrane (Amersham). For Southern blots, 7.5 μg of genomic DNA prepared from chicken embryos was digested with restriction enzymes EcoRI, BamHI, H
Cleavage with indIII and SacI and size fractionation on a 0.6% agarose gel. After electrophoresis, run the gel with 0.5 N NaOH
And denaturation in 1.5 M NaCl (30 min)
Neutralized in Tris-HCl (pH 7.5) and 1.5 M NaCl (30 min), the DNA was transferred onto a Hybond N + nylon membrane. Both Northern and Southern filters are 50% formamide, 5x SSC, 5x Denhard solution, 0.5% SDS
And 10 μg / ml denatured salmon sperm DNA, prehybridized at 37 ° C. for 1 hour, and then ³² P-radiolabeled DN under the same conditions as prehybridization for 12 hours.
Hybridized with the A probe. As a probe, a 0.6 kb EcoRI cDNA insert, λCEB201, labeled with a multi-prime labeling system (Amersham) was used. Filter with 2 x SSC and 0.1% SDS
Wash twice at 65 ° C. for 10 minutes in 0.2 × SSC and 0.
After washing twice in 1% SDS at 65 ° C. for 30 minutes, Kodak XAR film was exposed at −70 ° C. for about one day.

The amino acid sequence of the sialyltransferase P-B1 of the present invention elucidated as described above has the following characteristics which are not observed in the amino acid sequence of a conventionally known sialyltransferase. (i) All sialyltransferases that have been previously cloned are II
Type membrane proteins, which have a domain structure similar to other glycosyl-transferases. That is, NH
_Two- terminal short middle cytoplasmic tail, hydrophobic signal anchor domain, proteolytically sensitive stem region, and C
It has a large active domain at the OOH-terminus. On the other hand, the sialyltransferase P-B1 of the present invention has a large stem region (or an intermediate region).

(Ii) The sialyltransferase P-B1 of the present invention comprises:
It has a PEST region (residues 233-258). The amino acid sequence of a protein having an intracellular half-life of less than 2 hours has a region containing many proline, glutamic acid, serine, and threonine residues (PEST
Called: Rogers, S. et al., Science, 234, 364-3
68, 1986). These PEST regions generally flank a population containing several positively charged amino acids. Conventionally known sialyltransferases do not have this region. (iii) Two regions consisting of 8 amino acids (SSSXVSTC), 24
Found at residues 7-254 and 330-337. Searches of the Genebank database for other proteins did not reveal sequences similar to this sequence.

Previously known sialyltransferases showed surprisingly tissue-specific expression, which is thought to correlate with the presence of cell type-specific sugar chain structures (Paulson , JC and Colley, KJ, J. Biol. Chem.,
264, 17615-17618, 1989). According to the results of Northern blotting described above, the mRN of sialyltransferase P-B1
It is clear that the expression pattern of A changes. Three different sizes of sialyltransferase P-B1 genes
Transcription of mRNA (4.0, 3.0 and 2.2 kb) indicates that they are Gal β1,4GlcNAc-α2,6-sialyltransferase ( Gal
β4GlcNAc-α6STRL) and Gal β1,3 (4) GlcNAcα2,3-sialyltransferase ( Gal β3 (4) GlcNAc-α3STRL) (Weinstei
n, J. et al., J. Biol. Chem., 262, 17735-17743, 19
87; and Wen, DX et al., J. Biol. Chem., 267,
21011-21019, 1992), suggesting expression through an alternative splicing mechanism and an alternative promoter utilization mechanism. This is evident from the Southern hybridization showing that only a single copy of the gene exists for sialyltransferase P-B1.

GalNAcα2,6-sialyltransferase P of the present invention
-B1 remains in the cell after expression and is not secreted out of the cell, and when the intracellular concentration exceeds a certain level, the expression of the enzyme may decrease. Since the GalNAcα2,6-sialyltransferase P-B1 of the present invention utilizes the GalNAcα2,6-sialyltransferase activity of P-B1, the soluble form of the protein SB which is maintained in the activity of the enzyme and secreted from cells during expression -690 was manufactured.
The gene encoding the protein SB-690 of the present invention and the amino acid sequence of the protein SB-690 of the present invention are shown in SEQ ID NO: 2 in the sequence listing.

The sialyltransferase cloned so far has the same domain structure as other glycosyl-transferases. That is, a short cytoplasmic middle tail at the NH ₂ terminus, a hydrophobic signal anchor domain,
Proteolytically sensitive stem region, and COOH-
It has a large active domain at the end (Paulson, JC and
Colley, KJ, J. Biol. Chem., 264, 17615-17618, 1
989). In order to investigate the location of the transmembrane domain of the GalNAcα2,6-sialyltransferase of the present invention, the hydrophobicity distribution map was determined using the kite and J. and Doolittle, RF, J. M.
ol. Biol., 157, 105-132, 1982) (FIG. 2). As a result, the Ga
It was suggested that the hydrophobic transmembrane domain of lNAcα2,6-sialyltransferase P-B1 is 21 amino acid residues from amino acids 17 to 37.

As described above, the hydrophobic signal anchor domain of GalNAcα2,6-sialyltransferase is composed of amino acid residues
Located at 17-37. Although the in vitro translation / transcription system containing pSB-BGL as a template synthesizes a protein (33 kDa), the medium from cells transfected with pcDSB-BGL has no significant activity.
It was evident that 3 to 269 residues contained some essential residues for enzyme activity, and the active domain was estimated around 233-566 (FIG. 3). It is of a size comparable to that of other cloned sialyltransferases. To make a soluble protein containing the above active domain, P-B1
The sequence corresponding to the putative active domain was fused in-frame to the immunoglobulin signal peptide sequence (Joblin
g, SA and Gehrke, L., Nature (Lond.), 325, 622-62
5, 1987). The details of the experiment are shown below.

The vector plasmid pUGS was replaced with pBluescrip
t PSK-XhoI fragment of SK (+) plasmid
It was prepared by substituting the synthetic DNA fragment of p. This fragment is a 43 bp alfalfa mosaic virus with a synthetic PstI site at the 5 'end (Jobling,
SA and Gehrke, L., Nature (Lond.) 325, 622-625,19
87) contains a 5'-untranslated leader sequence, which contains a 17 bp synthetic EcoRI, B
Mouse immunoglobulin M heavy chain signal peptide sequence (57 bp) with glII and XhoI cloning sites (Boersch-
Supan, ME etal., J. Exp. Med. 161, 1272-1292, 19
85) is continued. The nucleotide sequence of this fragment is 5'-CTGCAGGGTTTTTATTTTTAATTTTCTTTCAAATACTTCCA
CCATGAAATTCAGCTGGGTCATGTTCTTCCTGATGGCAGTGGTTACAGGG
GTCAATTCAGAATTCCAGATCTCGAG-3 '.

The gene encoding GalNAcα2,6-sialyltransferase of the present invention, λCEB-3043, was partially digested with EcoRV, and the 1.8 kb fragment was digested with EcoRV of pBluescript SK (+).
Subcloning into the V site produced pCEB-1800. This clone lacks the 0.8 kb 3'-untranslated region of λCEB-3043. GalNAcα2,6-sialyltransferase
5'-AGGGCTGCTGAATTC with a synthetic EcoRI site in the center using the active domain of P-B1 as the 5'-primer
ACTGAGCCACAG-3 '(nucleotides 679-708) and 3'
Produced by PCR using universal M13 sequencing primers and pCEB-1800 as template. This PCR product was digested with EcoRI and XhoI, and ligated to the EcoRI / XhoI site of the vector plasmid pUGS to obtain plasmid pSB-690. This plasmid contains λCEB-30
Among the 34 genes, a sequence containing the region encoding the active domain of GalNAcα2,6-sialyltransferase P-B1 fused in-frame to the 3 ′ end of the immunoglobulin signal sequence is included. This fusion fragment was digested with PstI and XhoI.
After cutting from pSB-690 with pcDSRα expression vector,
Insertion into the PstI / XhoI site yielded pcDSB-690.

As a control, SB-BGL, a protein lacking a part of the active domain of GalNAcα2,6-sialyltransferase P-B1,
Was produced as follows. pCEB-1800 and pUGS to BglII
And sticky ends to DNA polymerase Klenow (Kle
Now) filled with fragments. The Klenow fragment of DNA polymerase was heat denatured (94
(C, 20 minutes), these plasmids were cut with XhoI. The 1.0 kb fragment from pCEB-1800 was gel purified and subcloned into the blunt-ended BglII / XhoI site of pUGS to yield plasmid pSB-BGL. The PstI / XhoI fragment from pSB-BGL was
Subcloning in the PstI / XhoI site yielded pcDSB-BGL.

The expression of the above proteins was carried out as follows.
COS-7 cells were treated with the DEAE-dextran method (McCutchan, JH a
nd Pagano, JSJ Natl. Cancer Inst. 41, 351-357,
1968) using 5 μg of plasmid DNA. After 48 hours of transfection, the medium was harvested and concentrated 10-fold using a Centricon 30 filter (Amicon) for enzyme assays. For metabolic labeling, COS cells (60-mm culture dishes) were washed with Met-free medium (Dulbecco's modified Eagle's medium and 2% fetal bovine serum) (GIBCO) and then incubated in the same medium for 1 hour. Cells in 1.5 ml Met-free medium
Pulse labeled with 10 MBq / dish of Express ³⁵ S Protein Labeling Mix (Dupont-New England Nuclear) for 2 hours. These cells are then
-Washed with free medium and chased for 5 hours in medium without express label. The medium containing the secreted protein was collected, concentrated 10-fold, and then subjected to SDS-polyacrylamide gel electrophoresis and then to fluorography.

The enzyme activity of the expressed protein was measured as follows. Assays using oligosaccharides and glycoproteins as acceptors in a total volume of 10 μl
50 mM sodium cacodylate buffer, pH 6.
0, 50 μM CMP- [ ¹⁴ C-] NeuAc (0.9 Bq / pmol), 1 mg / ml
Bovine serum albumin, 2 mg / ml acceptor substrate and 1
Performed in the presence of μl concentrated COS cell medium and incubated at 30 ° C. for 2 hours. At the end of the incubation period, 1 μl of the assay mixture was placed on a silica gel 60 HPTLC plate (Merck, Germany). This plate is ethanol: pyridine: n-butanol: water: acetic acid (100: 10: 10: 3
0: 3), and the radioactivity was visualized and quantified using a BAS2000 radio image analyzer (Fuji Photo Film, Japan). Radioactivity remaining at the origin was considered sialylated glycoprotein.

The sialylated product was identified as follows. Asialo-BSM was added to CMP- [
Re-sialylation and reduction with ¹⁴ C] NeuAc produced β-removed oligosaccharides. β-removal was performed by Carlson's method (C
arlson, DM, J. Biol. Chem., 243, 616-626, 1968)
Was performed according to Asialo-BSM (100 μg each) was treated with CMP- [ ¹⁴ C-] NeuA in pcDSB-690 COS cell medium under the same conditions as above, except that the incubation time was 12 hours.
Sialized with c. The reaction was stopped by adding 500 μl of 1% phosphotungstic acid in 0.5 M HCl and then centrifuged at 10,000 × g for 5 minutes. After washing the pellet once with the same phosphotungstic acid solution and once with methanol,
It was dissolved in 0.05 M NaOH and 1M NaBH ₄ solution in 0.5 ml, 45
Incubated for 30 hours at ° C.

At the end of the incubation period, the solution was neutralized to pH 6 with acetic acid and lyophilized. The dried product was dissolved in 50 μl of water and desalted by gel filtration on a Sephadex G-15 column (0.5 × 5 cm) equilibrating and eluting with water. The radioactive fraction was subjected to thin layer chromatography for product identification without further purification. Using two different developing solvents, N from natural BSM
euAcα2,6GalNAc-ol and GlcNAcβ1,3 [NeuAcα2,6]
Co-electrophoresed with GalNAc-ol. The ratio of transferred sialyl residues was 1: 0.9: 0.6. Sialylated GalNAc-SerNAc
NeuAcα2,6GalNA in two different developing solvent systems
By co-electrophoresis with c-SerNAc, the protein SB-
690 has been shown to form NeuAcα2,6 bonds to GalNAc which is directly linked to Ser or Thr residues in glycoproteins.

Sialyltransferase activity was detected in the medium in which cells transfected with pcDSB-690 were cultured.
It was revealed that the protein SB-690 of the present invention expressed by pcDSB-690 was secreted extracellularly while retaining sialyltransferase activity. On the other hand, sialyltransferase activity was not detected in the medium in which cells transfected with cDSB-BGL were cultured.

The receptor specificity of the protein SB-690 of the present invention
COS-7 cell cultures transfected with cDSB-690 were examined in concentrated culture medium. As shown in Table 1, asialomutin, fetuin and asialofetuin acted as good receptors. Remarkably, fetuin was shown to be a better receptor than asialofetuin (Baubichon-Cortay, H. et al., Carbohydr. Res., 1
49, 209-223, 1986; and Brockhausen, I. et al.,
Biochemistry, 29, 10206-10212, 1990). Other glycoproteins, oligosaccharides and glycolipids
Except for GalNAc-SerNAc, it did not act as an acceptor. These data indicate that the acceptor site may be associated with Ser or T in glycoprotein through α-glycosidic bonds.
This suggests that GalNAc is directly bound to the hr residue.

[0040]

[Table 1] Receptor specificity of protein SB-690 of the present invention ─ Receptor pmoles / hr / 10μl medium ─────────────────────────────────── Fetuin 142 Asialo-fetuin 96 α1 acid glycoprotein 6 asialo-α1 acid glycoprotein 4 bovine mandibular mucin 15 bovine mandibular asialomucin 186 egg mucoid 7 asialo-egg mucoid 0 Gal β1,3GlcNAc β1,3Galβ1,4Glc 0 Gal β1, 4GlcNAc 0 Gal β1,3GalNAc 0 GalNAcβ1,4Gal 0 Gal β1,4Glc 0 Galactose 0 Ganglioside mixture 0 Ganglioside GD1a 0 GalNAc-SerNAc 4 Benzyl-GalNAc 2 ──────────────────中 In the table, "0" indicates less than 1 pmole / hr / 10μl medium.

A conventionally cloned sialyltransferase shows acceptor specificity only for the Gal- moiety. On the other hand, the GalNAcα2,6-sialyltransferase P-B1 of the present invention and the protein SB-690 of the present invention show acceptor specificity for the GalNAc-part, but show acceptor specificity for the Gal-part. Not something. The following findings indicate that GalNAcα2,6-sialyltransferase PB
1 and the protein SB-690 of the present invention have an α2,6-linkage
FIG. 4 shows that Gal-NeuAc has a GalNAc α2,6-sialyltransferase activity of transferring CMP-NeuAc onto a GalNAc-residue O-linked to Thr / Ser of glycoprotein.

(I) Expression of pcDSB-690 in COS cells revealed acceptor specificity only for the GalNAc portion bound to Thr / Ser residues. On the other hand, no detectable enzyme activity was observed for the other substrates tested (Table 1). (ii) Bovine mandibular asialo-mucin and GalNAc-SerNAc
Sialylated product obtained from GalNAc through α2,6 linkage
It was shown to have sialic acid attached to the moiety. There are two types: bovine mandibular gland type and liver (brain) type
GalNAc-α6ST has been reported and has different acceptor specificities (Bergh, ME et al., J. Biol.
Chem., 258, 7430-7436, 1983). The former enzyme is GalNAc,
It has broad specificity for Galβ1,3GalNAc and NeuAcα2,3Galβ1,3GalNAc, while the latter has specificity only for the NeuAcα2,3Galβ1,3GalNAc portion of glycoprotein. GalNAcα2,6-sialyltransferase P-B1 of the present invention
And the acceptor specificity of the protein SB-690 of the present invention is:
It shows that it is similar to the former enzyme.

Examination of the acceptor site of asialomucin showed that NeuAcα2,6GalNAc-Ser / Thr was the most abundant product. However, the ratio of glycoconjugates in bovine mandibular gland asialomucin, i.e., GalNAc-Ser / Thr,
GlcNAcβ1,3GalNAc-Ser / Thr and Galβ1,3GalNAc-Ser / T
hr is 65%, 25% and 5% respectively (Tsuji,
T. and Osawa, T., Carbohydr. Res., 151, 391-402,
1986), the GalNAcα2,6-sialyltransferase P-B1 of the invention and the protein SB-690 of the invention have the following acceptor priority: Galβ1,3Gal
NAc-Ser / Thr>) GlcNAcβ1,3GalNAc-Ser / Thr> GalNA
c-Ser / Thr. On the other hand, the fact that almost all radioactivity is dissociated by weak alkaline treatment, and fetuin is more preferable than asialofetuin (Table 1)
Bovine liver (Bergh, ME et al., J. Biol. Chem., 258,
7430-7436, 1983) and rat brain (Baubichon-Cortay, H.
et al., Carbohydr. Res., 149, 209-223, 1986).
As reported for Acα2,6-sialyltransferase, NeuAcα2,3Galβ1,3GalNAc-Ser / Thr was replaced by Galβ1,3G
This indicates that the substrate is more preferable than alNAc-Ser / Thr.

The sialylation of GalNAc-SerNAc is much slower than the sialylation of the corresponding residue on asialomtin (Table 1). Brockhausen et al., Bioch
emistry, 29, 10206-10212, 1990), a minimum length of 5 amino acids is required for effective synthetase activity. It is likely that the GalNAcα2,6-sialyltransferase P-B1 of the present invention and the protein SB-690 of the present invention also have a similar direct action on the peptide portion (Table 1).

The reagents and samples used in the above experiments and the detailed experimental methods are as follows. Fetuin, asialofetuin, bovine mandibular gland mucin, α1-acid glycoprotein, galactose β1,4-N-acetylgalactosamine, CMP-NeuAc, lacto-N-tetraose, benzyl-GalNAc, N-acetyllactosamine, and Triton CF -54 was obtained from Sigma (St. Louis, USA). CMP- [ ¹⁴ C] NeuAc (11 GBq / mmole) was purchased from Amersham (U.S.A.).
K.). N-acetylgalactosamine β1,4-galactose used was donated by Dr. Kajimoto (RIKEN, Wako, Saitama). 2-acetamide, 2-deoxygalactosyl αN-acetylserine (GalNA
c-SerNAc) is Grandler and Schmidt (Grundler
G., and Schmidt RR, Liebigs Ann. Chem., 1984, 18
26-1847, 1984).

NeuAc α2,6-GalNAc-SerNAc is NeuAcα2,
Prepared from 6GalNAc-Ser (Mect) by acetylation with acetic anhydride in pyridine-water. NeuAcα2,6GalNAc-ol and GlcNAcβ1,3 [NeuAcα2,6] GalNAc-ol were obtained from Tsuji, T. and Osawa T., Carbohydr. Re.
, 151, 391-402, 1986) and identified by 270 MHz ¹ H and ¹³ C NMR (Savage, AV et al., Eur. J. Biochem., 192, 427).
-432, 1990; and Savage, AV et al., Eur. J. Bio
chem., 193, 837-843, 1990). Synthetic primers were synthesized on an Applied Biosystems 394 DNA synthesizer. Restriction enzymes SmaI, EcoRI, BamHI, HindIII, Sa
cI, XhoI, BglII and PstI were obtained from Takara Shuzo.

[0047]

According to the present invention, a novel enzyme P-B1 and a novel protein SB- having an active part of the enzyme and secreted extracellularly
690 is provided. The enzyme and protein of the present invention are GalNAcα.
Since it has 2,6-sialyltransferase activity, it is useful, for example, as a reagent for introducing a human-type sugar chain into a protein. In addition, it is useful as a medicament for treating a hereditary disease lacking a human-specific sugar chain. Furthermore, it can be used as a medicine for suppressing cancer metastasis, preventing viral infection, and suppressing inflammatory response.

[0048]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 2072 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin: G. gallus (chicken) Sequence characteristics: CDS 1-1698 sequence (-31) CCGAGCTTCCATCTCTCCCGGGCCTCTCACT -1 ATG GGG TTT TTA ATC AGA AGG CTT CCT AAA GAT TCC AGA ATA TTC 45 MET Gly Phe Leu Ile Arg Arg Leu Pro Lys Asp Ser Arg Ile Phe 15 CGT TGG CTC CTT ATT TTA TCA TTC TTC ATC ATT ACT AGT 90 Arg Trp Leu Leu Ile Leu Thr Val Phe Ser Phe Ile Ile Thr Ser 30 TTT AGC GCC TTG TTT GGC ATG GAG AAA AGC ATT TTC AGG CAG CTC 135 Phe Ser Ala Leu Phe Gly MET Glu Lys Ser Ile Phe Arg Gln Leu 45 AAG ATT TAC CAA AGC ATT GCA CAT ATG CTA CAA GTG GAC ACC CAA 180 Lys Ile Tyr Gln Ser Ile Ala His MET Leu Gln Val Asp Thr Gln 60 GAT CAG CAA GGT TCA AAC TAT TCT GCT AAT GGG AGA ATT TCA AAG 225 Asp Gln Gln Gly Ser Asn Tyr Ser Ala Asn Gly Arg Ile Ser Lys 75 GTT GGT TTG GAG AGA GAC ATT GCA TGG CTC GAA CTG AAT ACT GCT 270 Val Gly Leu Glu Arg Asp Ile Ala Trp Leu Glu Leu Asn Thr Ala 90 GTG AGT ACA CCA AGT GGG GAA GGG AAG GAA GAG CAG AAG AAA ACA 315 Val Ser Thr Pro Ser Gly Glu Gly Lys Glu Glu Gln Lys Lys Thr 105 GTG AAA CCA GTT GCC AAG GTG GAA GAA GCC AAG GAG AAA GTG ACT 360 Val Lys Pro Val Ala Lys Val Glu Glu Ala Lys Glu Lys Val Thr 120 GTG AAA CCA TTC CCT GAG GTG ATG GGG ATC ACA AAT ACA ACA GCA 405 Val Lys Pro Phe Pro Glu Val MET Gly Ile Thr Asn Thr Thr Ala 135 TCA ACA GCC TCT GTG GTG GAG AGA ACA AAG GAG AAA ACA ACA GCG 450 Ser Thr Ala Ser Val Val Glu Arg Thr Lys Glu Lys Thr Thr Ala 150 AGA CCA GTT CCA GGG GTG GGG GAA GCT GAT GGG AAG AGA ACA ACG 495 Arg Pro Val Pro Gly Val Gly Glu Ala Asp Gly Lys Arg Thr Thr 165 ATA GCA CTT CCC AGC ATG AAG GAA GAC AAA GAG AAG GCG ACT GTG 540 Ile Ala Leu Pro Ser MET Lys Glu Asp Lys Glu Lys Ala Thr Val 180 AAA CCA TCC TTT GGG ATG AAG GTA GCT CAT GCA AAC AGC ACA TCC 585 Lys Pro Ser Phe Gly MET Lys Val Ala His Ala Asn Ser Thr Ser 195 AAA GAT AAA CCA AAG GCA GAA GAG CCT CCT GCA TCA GTG AAA GCC 630 Lys Asp Lys Pro Lys Ala Glu Glu Pro Pro Ala Ser Val Lys Ala 210 ATA AGA CCT GTG ACT CAG GCT GCC ACA GTG ACA GAG AAG AAG AAA 675 Ile Arg Pro Val Thr Gln Ala Ala Thr Val Thr Glu Lys Lys Lys 225 CTG AGG GCT GCT GAC TTC AAG ACT GAG CCA CAG TGG GAT TTT GAT 720 Leu Arg Ala Ala Asp Phe Lys Thr Glu Pro Gln Trp Asp Phe Asp 240 GAT GAG TAC ATA CTG GAT AGC TCA TCT CCA GTA TCG ACC TGC TCT 765 Asp Glu Tyr Ile Leu Asp Ser Ser Ser Pro Val Ser Thr Cys Ser 255 GAA TCA GTG AGA GCC AAG GCT GCC AAG TCT GAC TGG CTG CGA GAT 810 Glu Ser Val Arg Ala Lys Ala Ala Lys Ser Asp Trp Leu Arg Asp 270 CTT TTC CTG CCG AAC ATC ACA CTC TTC ATA GAC AAG AGT TAC TTC 855 Leu Phe Leu Pro Asn Ile Thr Leu Phe Ile Asp Lys Ser Tyr Phe 285 AAT GTC AGT GAG TGG GAC CGC CTG GAG CAT TTT GCA CCT CCC TAT 900 Asn Val Ser Glu Trp Asp Arg Leu Glu His Phe Ala Pro Pro Tyr 300 GGC TTC ATG GAG CTG AAT TAC TCA CTG GTA GAA GAA GTC ATG TCA 945 Gly Phe MET Glu Leu Asn Tyr Ser Leu Val Glu Glu Val MET Ser 315 CGG CTG CCT CCA AAT CCC CAC CAG CAG CTG CTC CTG GCC AAC AGT 990 Arg Leu Pro Pro Asn Pro His Gln Gln Leu Leu Leu Ala Asn Ser 330 AGC AGC AAC GTG TCA ACG TGC ATC AGC TGT GCT GTT GTG GGG AAT 1035 Ser Ser Asn Val Ser Thr Cys Ile Ser Cys Ala Val Val Gly Asn 345 GGA GGG ATA TTG AAT AAC TCT GGA ATG GGC CAG GAG ATT GAC TCC 1080 Gly Gly Ile Leu Asn Asn Ser Gly MET Gly Gln Glu Ile Asp Ser 360 CAT GAC TAT GTG TTC CGG GTG AGC GGG GCT GTA ATC AAA GGT TAC 1125 His Asp Tyr Val Phe Arg Val Ser Gly Ala Val Ile Lys Gly Tyr 375 GAA AAG GAT GTG GGA ACA AAA ACC TCC TTC TAC GGA TTC ACA GCG 1170 Glu Lys Asp Val Gly Thr Lys Thr Ser Phe Tyr Gly Phe Thr Ala 390 TAC TCC TCC CTG GTG TCC TCT CTC CAG AAC TTG GGA CAC AAA GGG TTC 1215 Tyr Ser Leu Val Ser Ser Leu Gln Asn Leu Gly His Lys Gly Phe 405 AAG AAG ATC CCA CAG GGG AAG CAT ATC AGA TAC ATT CAC TTC CTG 1260 Lys Lys Ile Pro Gln Gly Lys His Ile Arg Tyr Ile His Phe Leu 420 GAG GCA GTT AGA GAC TAT GAG TGG CTG AAG GCT CTT CTG TTG GAC 1305 Glu Ala Val Arg Asp Tyr Glu Trp Leu Lys Ala Leu Leu Leu Asp 435 AAG GAT ATC AGG AAA GGA TTC CTG AAC TAC TAT GGG CGA AGG CCC 1350 Lys Asp Ile Arg Lys Gly Phe Leu Asn Tyr Tyr Gly Arg Arg Pro 450 CGG GAG AGA TTC GAT GAA GAT TTC ACA ATG AAT AAG TAC CTG GTA 1395 Arg Glu Arg Phe Asp Glu Asp Phe Thr MET Asn Lys Tyr Leu Val 465 GCT CAC CCT GAT TTC CTC AGA TAC TTG AAA AAC AGG TTC TTA AAA 1440 Ala His Pro Asp Phe Leu Arg Tyr Leu Lys Asn Arg Phe Leu Lys 480 TCT AAA AAT CTG CAA AAG CCC TAC TGG CGG CTG TAC AGA CCC ACA 1485 Ser Lys Asn Leu Gln Lys Pro Tyr Trp Arg Leu Tyr Arg Pro Thr 495 ACA GGA GCC CTC CTG CTG CTG ACT GCC CTG CAT CTC TGT GAC CGG 1530 Thr Gly Ala Leu Leu Leu Leu Thr Ala Leu His Leu Cys Asp Arg 510 GTG AGT GCC TAT GGC TAC ATC ACA GAA GGT CAC CAG AAG TAC TCG 1575 Val Ser Ala Tyr Gly Tyr Ile Thr Glu Gly His Gln Lys Tyr Ser 525 GAT CAC TAC TAT GAC AAG GAG TGG AAA CGC CTG GTC TTC TAC GTT 1620 Asp His Tyr Tyr Asp Lys Glu Trp Lys Arg Leu Val Phe Tyr Val 540 AAC CAT GAC TTC AAC TTG GAG AAG CAG GTG TGG AAA AGG CTT CAT 1665 Asn His Asp Phe Asn Leu Glu Lys Gln Val Trp Lys Arg Leu His 555 GAT GAG AAC ATC ATG AAG CTC TAC CAG AGA TCC TGA CAG TGT GCC 1710 Asp Glu Asn Ile MET Lys Leu Tyr Gln Arg Ser --- 566 GAGGGCCATT GCCTGGGAAA TCTCAACAGC ACCTCATGGG GAACAGAAGA 1760 GGACCTCGGA AGCCAGGGTT AGCTCTGGAC TTCCAGGCCC AGCTTCAGCT 1810 CCACAGAGAT ATTTCCCTCC TTTGATATCT TTATTTTCTC ACAACACTTC 1860 CTAAAATGTG CATATTCTAC AGACCAAGCG AACAGTAGGG AAAAGTGCCT 1910 CCAAACAAGG TCCCATCTGA CTTGTGGACG GTTGTAGGCT CTGGTACTGG 1960 GAAAGAGGAA TCCGGGATGA ATCCGAATAG CAGATGTTCC AGTGCCCATT 2010 ATCTTAATCA GGTTCTCCCT CTGCAAGGAG ATGCTCTTGG GGCTGGGGCT 2060 AGTTTTGCTC TAGGTGGGTT CTCTCTGTGA GTAGTGCTTG TTATGGAGCT 2110 GGGTGTTTTG GGTAAGCAGT GGATAGAATG GAGACACACA CAATCCTGTC 2160 TCAAGAGGAT GATTTGTGTC CTGGAGGTGC TGCTGTCACT CTGCTCACTG 2210 CAGGCATAAG GACCCTTCCA ATGAACTCAA TCCCAATGTG ACTTTGCTGT 2260 GACACCTCCT GGGGAGCACT GTGATGTCGG TGCCCAGCCT GCTGCCCTTG 2310 GCCTAGTTCA CCATCAGCAC AAGGGAAGGG GAGAGCCCTC CGTAGTGCAG 2360 CAGAATGCTG GACATTGTAC CTCTTGCTGT GGGTTCCCCT GGCTGCAGAC 2410 TACGTGTAGT GAGTCTGATG AAGAAGCTGG TGCTTG GCTG TGCCAGGAGC 2460 ATGGTGCTTC CTCTTCTACC AGGAGAAATG AGAATTCTCA ATGTCCATGG 2510 ATGGATGCTG TCTGTCCTTG CTGCTGGCTG GAGTGCCTGC CTACATTGTC 2560 CTGAGAAAAG CACTGTTACA GCCAGTAAGC CTTTGGAGTA TTGGCCTTCT 2610 GAGTGGGCTT TTGCAAACAA AATAAACGGC ACTGCTTTCC CCCAAGCTGA 2660 AAAAAAAAAA A 2671 SEQ ID NO: 2 sequence Length: type 1294 sequence: the number of a nucleic acid strand: 2 Main chain topology: Linear Sequence type: cDNA to mRNA Origin: G. gallus (chicken) Sequence characteristics: CDS 1-1065 Sequence characteristics: signal peptide 1-1065 sequence (-43) CTG CAGGGTTTTT ATTTTTAATT TTCTTTCAAA TACTTCCACC -1 PstI ATG AAA TTC AGC TGG GTC ATG TTC TTC CTG ATG GCA GTG GTT ACA 45 MET Lys Phe Ser Trp Val MET Phe Phe Leu MET Ala Val Val Thr 15 GGG GTC AAT TCA GAA TTC ACT GAG CCA CAG TGG GAT TTT GAT GAT 90 Gly Val Asn Ser Glu Phe Thr Glu Pro Gln Trp Asp Phe Asp Asp 30 GAG TAC ATA CTG GAT AGC TCA TCT CCA GTA TCG ACC TGC TCT GAA 135 Glu Tyr Ile Leu Asp Ser Ser Ser Pro Val Ser Thr Cys Ser Glu 45 TCA GTG AGA GCC AAG GCT GCC AAG TCT GAC TGG CTG CGA GAT CTT 180 Ser Val Arg Ala Lys Ala Ala Lys Ser Asp Trp Leu Arg Asp Leu 60 TTC CTG CCG AAC ATC ACA CTC TTC ATA GAC AAG AGT TAC TTC AAT 225 Phe Leu Pro Asn Ile Thr Leu Phe Ile Asp Lys Ser Tyr Phe Asn 75 GTC AGT GAG TGG GAC CGC CTG GAG CAT TTT GCA CCT CCC TAT GGC 270 Val Ser Glu Trp Asp Arg Leu Glu His Phe Ala Pro Pro Tyr Gly 90 TTC ATG GAG CTG AAT TAC TCA CTG GTA GAA GAA GTC ATG TCA CGG 315 Phe MET Glu Leu Asn Tyr Ser Leu Val Glu Glu Val MET Ser Arg 105 CTG CCT CCA AAT CCC CAC CAG CAG CTG CTC CTG GCC AAC AGT AGC 360 Leu Pro Pro Asn Pro His Gln Gln Leu Leu Leu Leu Ala Asn Ser Ser 120 AGC AAC GTG TCA ACG TGC ATC AGC TGT GCT GTT GTG GGG AAT GGA 405 Ser Asn Val Ser Thr Cys Ile Ser Cys Ala Val Val Gly Asn Gly 135 GGG ATA TTG AAT AAC TCT GGA ATG GGC CAG GAG ATT GAC TCC CAT 450 Gly Ile Leu Asn Asn Ser Gly MET Gly Gln Glu Ile Asp Ser His 150 GAC TAT GTG TTC CGG GTG AGC GGG GCT GTA ATC AAA GGT TAC GAA 495 Asp Tyr Val Phe Arg Val Ser Gly Ala Val Ile Lys Gly Tyr Glu 165 AAG GAT GTG GGA ACA AAA ACC TCC TTC TAC GGA TTC ACA GCG TAC 540 Lys Asp Val Gly Thr Lys Thr Ser Phe Tyr Gly Phe Thr Ala Tyr 180 TCC CTG GTG TCC TCT CTC CAG AAC TTG GGA CAC AAA GGG TTC AAG 585 Ser Leu Val Ser Ser Leu Gln Asn Leu Gly His Lys Gly Phe Lys 195 AAG ATC CCA CAG GGG AAG CAT ATC AGA TAC ATT CAC TTC CTG GAG 630 Lys Ile Pro Gln Gly Lys His Ile Arg Tyr Ile His Phe Leu Glu 210 GCA GTT AGA GAC TAT GAG TGG CTG AAG GCT CTT CTG TTG GAC AAG 675 Ala Val Arg Asp Tyr Glu Trp Leu Lys Ala Leu Leu Leu Asp Lys 225 GAT ATC AGG AAA GGA TTC CTG AAC TAC TAT GGG CGA AGG CCC CGG 720 Asp Ile Arg Lys Gly Phe Leu Asn Tyr Tyr Gly Arg Arg Pro Arg 240 GAG AGA TTC GAT GAA GAT TTC ACA ATG AAT AAG TAC CTG GTA GCT 765 Glu Arg Phe Asp Glu Asp Phe Thr MET Asn Lys Tyr Leu Val Ala 255 CAC CCT GAT TTC CTC AGA TAC TTG AAA AAC AGG TTC TTA AAA TCT 810 His Pro Asp Phe Leu Arg Tyr Leu Lys Asn Arg Phe Leu Lys Ser 270 AAA AAT CTG CAA AAG CCC TAC TGG CGG CTG TAC AGA CCC ACA ACA 855 Lys Asn Leu Gln Lys Pro Tyr Trp Arg Leu Tyr Arg Pro Thr Thr 285 GGA GCC CTC CTG CTG CTG ACT GCC CTG CAT CTC TGT GAC CGG GTG 900 Gly Ala Leu Leu Leu Leu Thr Ala Leu His Leu Cys Asp Arg Val 300 AGT GCC TAT GGC TAC ATC ACA GAA GGT CAC CAG AAG TAC TCG GAT 945 Ser Ala Tyr Gly Tyr Ile Thr Glu Gly His Gln Lys Tyr Ser Asp 315 CAC TAC TAT GAC AAG GAG TGG AAA CGC CTG GTC TTC TAC GTT AAC 990 His Tyr Tyr Asp Lys Glu Trp Lys Arg Leu Val Phe Tyr Val Asn 330 CAT GAC TTC AAC TTG GAG AAG CAG GTG TGG AAA AGG CTT CAT GAT 1035 His Asp Phe Asn Leu Glu Lys Gln Val Trp Lys Arg Leu His Asp 345 GAG AAC ATC ATG AAG CTC TAC CAG AGA TCC TGA CAGTGTGCCGAG 1080 Glu Asn I MET Lys Leu Tyr Gln Arg Ser --- 360 GGCCATTGCC TGGGAAATCT CAACAGCACC TCATGGGGAA CAGAAGAGGA 1175 CCTCGGAAGC CAGGGTTAGC TCTGGACTTC CAGGCCCAGC TTCAGCTCCA 1225 CAGAGATATT TCCCTCCTTT GATATC 1251 EcoRV

[Brief description of the drawings]

FIG. 1. GalNAcα2,6-sialyltransferase PB of the present invention
1 is a restriction map of a cDNA clone encoding 1. In the figure, E: EcoRI; RV: EcoRV; P: PstI; B: BglII.

FIG. 2 GalNAcα2,6-sialyltransferase PB of the present invention
FIG. 2 is a view showing the results of a hydrophobicity analysis of No. 1. In the figure, the left side is the protein N-terminal, and a positive value indicates a hydrophobic portion.

FIG. 3 GalNAcα2,6-sialyltransferase PB of the present invention
FIG. 2 is a view showing a comparison between the position of the active domain No. 1 and the protein SB-690 of the present invention. In the figure, protein SB-BGL is GalNAcα2,6-
This protein does not show sialyltransferase activity.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Li, Kiharu, 2-1 Hirosawa, Wako-shi, Saitama Pref. (72) Inventor Takashi Nakaoka 2-1 Hirosawa, Wako-shi, Saitama Pref. (72) Invention Naoya Kojima 2-1 Hirosawa, Wako-shi, Saitama Pref. RIKEN (56) References Biol. Chem. 268 (16) p. 11504-11507 (1993) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 9/10 C12N 15/54 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI (DIALOG)

Claims (15)

(57) [Claims] 1. A GalNAcα2,6-sialyltransferase P-B1 specified by the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 2. The GalNAc α2,6- according to claim 1.
GalN encoding the amino acid sequence of sialyltransferase
Acα2,6-sialyltransferase gene. 3. The nucleotide sequence according to claim 2, which has a nucleotide sequence specified by nucleic acid numbers 1 to 1698 shown in SEQ ID NO: 1 in the sequence listing.
The described GalNAcα2,6-sialyltransferase gene. 4. The GalNAc α2,6- according to claim 2,
A recombinant vector containing a sialyltransferase gene. 5. The recombinant vector according to claim 4, which is a plasmid λ CEB-3034. 6. A transformant transformed by the vector according to claim 4 or 5. 7. GalNAcα specified by the amino acid sequence 233 to 566 shown in SEQ ID NO: 1 in the sequence listing.
2,6-sialyltransferase active domain. 8. The GalNAcα2,6- according to claim 1.
An extracellular secretory protein containing a polypeptide portion, which is an active domain of sialyltransferase, and a signal peptide, which catalyzes GalNAcα2,6-sialyltransferase. 9. The protein SB-69 according to claim 8, which is specified by the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
0. 10. A gene encoding the protein according to claim 8 or 9. 11. The method according to claim 1, which has a nucleotide sequence represented by nucleic acid numbers 1 to 1065 shown in SEQ ID NO: 2 in the sequence listing.
0 described gene. A recombinant vector comprising the gene according to claim 10 or 11. 13. The recombinant vector according to claim 12, which is a plasmid pcDSB-690. A transformant transformed by the recombinant vector according to claim 12 or 13. 15. The method for producing a protein according to claim 8 or 9, wherein the transformant according to claim 14 is cultured, and the protein according to claim 8 or 9 is collected from the culture. .