U.S. patent application number 12/155320 was filed with the patent office on 2009-03-26 for glycosyltransferase, nucleic acid encoding the glycosyltransferase and method of testing canceration using the nucleic acid.
This patent application is currently assigned to National Institute of Advanced Industrial Science and Technology. Invention is credited to Toru Hiruma, Takashi Kudo, Hisashi Narimatsu, Akira Togayachi.
Application Number | 20090081668 12/155320 |
Document ID | / |
Family ID | 32708465 |
Filed Date | 2009-03-26 |
United States Patent
Application |
20090081668 |
Kind Code |
A1 |
Narimatsu; Hisashi ; et
al. |
March 26, 2009 |
Glycosyltransferase, nucleic acid encoding the glycosyltransferase
and method of testing canceration using the nucleic acid
Abstract
A tumor marker nucleic acid of the present invention is
concerned with a nucleic acid hybridizing under stringent
conditions to a nucleotide sequence described in SEQ ID NO: 1 or a
complementary nucleotide sequence thereof. A method of testing
canceration of the present invention is a method comprising
diagnosing a biological sample as being cancerous when the
transcription level of the nucleic acid in the biological sample
significantly exceeds that in a normal biological sample as a
control. The present invention also relates to a
.beta.1,3-N-acetyl-D-glucosaminyltransferase protein having an
activity of transferring N-acetyl-D-glucosamine from a donor
substrate to an acceptor substrate through .beta.1,3-linkage.
Inventors: |
Narimatsu; Hisashi;
(Tsukuba-shi, JP) ; Kudo; Takashi; (Tsukuba-shi,
JP) ; Togayachi; Akira; (Tsukuba-shi, JP) ;
Hiruma; Toru; (Tsukuba-shi, JP) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
National Institute of Advanced
Industrial Science and Technology
Tokyo
JP
|
Family ID: |
32708465 |
Appl. No.: |
12/155320 |
Filed: |
June 2, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10539834 |
Jan 30, 2006 |
7396666 |
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PCT/JP2003/017030 |
Dec 26, 2003 |
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12155320 |
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Current U.S.
Class: |
435/6.11 ;
435/320.1; 435/325; 435/6.18; 435/69.1; 530/387.9; 536/24.31 |
Current CPC
Class: |
C12N 9/1051
20130101 |
Class at
Publication: |
435/6 ;
536/24.31; 435/320.1; 435/325; 435/69.1; 530/387.9 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 15/11 20060101 C12N015/11; C12N 15/00 20060101
C12N015/00; C12N 5/06 20060101 C12N005/06; C12P 21/04 20060101
C12P021/04; C07K 16/18 20060101 C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 2002 |
JP |
380975/2002 |
Claims
1. A nucleic acid hybridizing under stringent conditions to a
nucleotide sequence described in SEQ ID NO: 1 or a complementary
nucleotide sequence thereof.
2. The nucleic acid according to claim 1, wherein the nucleic acid
consists of a nucleotide sequence having at least 15 contiguous
nucleotides in a nucleotide sequence described in SEQ ID NO: 1 or a
complementary nucleotide sequence thereof.
3. The nucleic acid according to claim 2, wherein the nucleic acid
consists of a nucleotide sequence described in SEQ ID NO: 1 or a
complementary nucleotide sequence thereof.
4. The nucleic acid according to claim 1, wherein the nucleic acid
is a probe or a primer.
5. The nucleic acid according to claim 1, wherein the nucleic acid
is a tumor marker.
6. A method of testing canceration of a biological sample,
comprising: (a) using a nucleic acid according to claim 1 to
measure the transcription level of the nucleic acid in the
biological sample; and (b) diagnosing the biological sample as
being cancerous when the transcription level of the nucleic acid in
the biological sample significantly exceeds that in a normal
biological sample as a control.
7. The method according to claim 6, wherein the biological sample
is a sample derived from the large intestine or peripheral
blood.
8. A method of testing canceration of a biological sample
comprising: (a) using a nucleic acid according to claim 1 as a
labeled probe, which is in turn brought into contact with the
biological sample under stringent hybridization conditions to
measure the transcription level of the nucleic acid in the
biological sample based on a signal from the label of the
hybridized nucleic acid; and (b) diagnosing the biological sample
as being cancerous when the transcription level of the nucleic acid
in the biological sample significantly exceeds that in a normal
biological sample as a control.
9. A method of testing canceration of a biological sample,
comprising: (a) using a primer according to claim 4 that is labeled
to subject a biological sample to nucleic acid amplification and
measuring the amount of a resulting nucleic acid amplification
product; and (b) diagnosing the biological sample as being
cancerous when the amount of the nucleic acid amplification product
significantly exceeds that in a normal biological sample as a
control.
10. The method according to claim 10, wherein the biological sample
is a sample derived from the large intestine or peripheral
blood.
11. A method of examining the effectiveness of treatment for cancer
therapy by use of a nucleic acid according to claim 1, comprising:
using the nucleic acid to measure the transcription level of the
nucleic acid in a biological sample that has received treatment for
cancer therapy and comparing its measurement value with that before
the treatment or without the treatment, thereby determining whether
the treatment given to the biological sample is effective or
not.
12. The method according to claim 11, comprising: using the
biological sample which has already been cancerous and determining
that treatment for cancer therapy given to the biological sample is
effective when the transcription level of the nucleic acid in the
biological sample that has received the treatment is significantly
below that before the treatment or without the treatment.
13. The method according to claim 11, wherein the biological sample
is an in vivo biological sample from a non-human model animal.
14. The method according to claim 11, wherein the biological sample
is a sample derived from the large intestine or peripheral
blood.
15. A nucleic acid comprises a nucleotide sequence from nucleotide
Nos. 97 to 1194 described in SEQ ID NO: 1 or a complementary
nucleotide sequence thereof.
16. The nucleic acid according to claim 15, wherein the nucleic
acid is DNA.
17. A vector comprising a nucleic acid according to claim 15.
18. A transformant comprising a vector according to claim 17.
19. A method of producing a
.beta.1,3-N-acetyl-D-glucosaminyltransferase protein, comprising:
growing a transformant according to claim 18 and expressing the
glycosyltransferase protein to collect the glycosyltransferase
protein from the transformant.
20. An antibody recognizing a
.beta.1,3-N-acetyl-D-glucosaminyltransferase protein comprising the
amino acid sequence of SEQ ID NO: 2 OR SEQ ID NO: 16.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel nucleic acid, the
nucleic acid for testing canceration, and a method of testing the
canceration of a biological sample based on a difference in the
expression level of the nucleic acid in the biological sample; as
well as, to a novel glycosyltransferase and a nucleic acid encoding
the glycosyltransferase, and the like.
BACKGROUND ART
[0002] In recent years, attention has been given to the function of
oligosaccharides and glycoconjugates in living bodies. For example,
a determinant factor of a blood type is a glycoprotein, and one
involved in the function of the nervous system is a glycolipid.
Thus, an enzyme having the function of synthesizing an
oligosaccharide is a crucially important key for analyzing
physiological activities produced by various oligosaccharides.
[0003] A N-acetyl-D-glucosamine residue (GlcNAc) and a D-galactose
residue (Gal), and the like, in sugar are the components of
glycosaminoglycan, while they are sugar residues present in various
oligosaccharide structures such as sphingoglycolipids, mucin-type
oligosaccharides, and asparagine-linked oligosaccharides (N-linked
oligosaccharides). Thus, an enzyme transferring GlcNAc or Gal is a
crucially important tool for analyzing the function of
oligosaccharides that work in various tissues in living bodies.
[0004] For example, at least 20 types of
N-acetylglucosaminyltransferases having an activity of transferring
GlcNAc have been known as shown in Table 1, each of which differs
in acceptor substrate specificity (References 1 to 18).
[0005] On the other hand, oligosaccharide synthesis is known to be
altered with great frequency in canceration and to be correlated
with the metastasis and malignancy of cancer (References 30 to 32).
Their comprehensive studies actively conducted today, for example,
analysis such as expression profiling in a variety of tissues, are
also directed to the elucidation of a canceration mechanism, and
discussions have often been conducted on the possibility that the
canceration mechanism is associated with the expression level of a
particular gene. As well known, the test of tumor markers or the
like in blood and the identification of the other gene products
involved in canceration, and so on, have already been conducted as
methods of cancer diagnostic tests. Tumor markers include many
antibodies against oligosaccharides. Among others, immunoassay for
oncogene products has often been adopted because of its advantage
in high sensitivity.
TABLE-US-00001 TABLE 1 N-acetylglucosaminyltransferases and their
substrate specificity Linkage Official name Abbreviation type
Substrate specificty Reference N-acetylglucosaminyltransferase-I
GnT-I .beta.1-2
Man.alpha.1-3(Man.alpha.1-6)Man.alpha.1-6(Man.alpha.1- 1
3)Man.beta.1-4GlcNAc.beta.1-4G3cNAc.beta.1-Asn
N-acetylglucosaminyltransferase-II GnT-II .beta.1-2
Man.alpha.1-6(GlcNAc.beta.1-2Man.alpha.1- 2
3)Man.beta.1-4GlcNAc.beta.1-4G3cNAc.beta.1-Asn
N-acetylglucosaminyltransferase-III GnT-III .beta.1-4
GlcNAc.beta.1-2Man.alpha.1-6(GlcNAc.beta.1- 3
2Man.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4G3cNAc.beta.1-Asn
N-acetylglucosaminyltransferase-IV GnT-IV .beta.1-4
GlcNAc.beta.1-2(GlcNAc.beta.1-6)Man.alpha.1- 4
6(GlcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-
4GlcNAc.beta.1-4G3cNAc.beta.1-Asn N-acetylglucosaminyltransferase-V
GnT-V .beta.1-6 GlcNAc.beta.1-2Man.alpha.1-6(GlcNAc.beta.1- 5
2(GlcNAc.beta.1-4)Man.alpha.1-3)Man.beta.1-
4GlcNAc.beta.1-4G3cNAc.beta.1-Asn
N-acetylglucosaminyltransferase-VI GnT-VI .beta.1-4
GlcNAc.beta.1-2(GlcNAc.beta.1-6)Man.alpha.1- 6
6(GlcNAc.beta.1-2(GlcNAc.beta.1-4)Man.alpha.1-3)Man.beta.1-
4GlcNAc.beta.1-4G3cNAc.beta.1-Asn
.beta.1,3-N-acetylglucosaminyltransferase IGnT .beta.1-3
Gal.beta.1-4GlcNAc.beta.1-R 7
.beta.1,3-N-acetylglucosaminyltransferase-2 .beta.3GnT2 .beta.1-3
Gal.beta.1-4GlcNAc.beta.1-R 8
.beta.1,3-N-acetylglucosaminyltransferase-3 .beta.3GnT3 .beta.1-3
Gal.beta.1-3GalNAc-0-S/T 8
.beta.1,3-N-acetylglucosaminyltransferase-4 .beta.3GnT4 .beta.1-3
Gal.beta.1-4(GlcNAc.beta.1-3Gal.beta.1-4)n-R 8
.beta.1,3-N-acetylglucosaminyltransferase-5 .beta.3GnT5 .beta.1-3
Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4-Cer 9
.beta.1,3-N-acetylglucosaminyltransferase-6 .beta.3GnT6 .beta.1-3
GalNAc-0-S/T 10 .beta.1,3-N-acetylglucosaminyltransferase-7
.beta.3GnT7 .beta.1-3
Gal.beta.1-4(GlcNAc.beta.1-3Gal.beta.1-4)n-Cer 11
.beta.1,3-N-acetylglucosaminyltransferase Fringe .beta.1-3
C2-X-X-G-G-(Fuc-0) S/T-C3 12
.beta.1,6-N-acetylglucosaminyltransferase IGnT .beta.1-6
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc.beta.1-R 13 Core 2 .beta.1,6-N-
C2GnT-I .beta.1-6 Gal.beta.1-3GalNAc-0-S/T 14
acetylglucosaminyltransferase-I Core 2 .beta.1,6-N- C2GnT-II
.beta.1-6 Gal.beta.1-3GalNAc-0-S/T 15
acetylglucosaminyltransferase-II Core 2 .beta.1,6-N- C2GnT-III
.beta.1-6 Gal.beta.1-3GalNAc-0-S/T 16
acetylglucosaminyltransferase-III .alpha.1,4-N- .alpha.4GnT
.alpha.1-4 Gal.beta.1-3(Gal.beta.1-4GlcNAc.beta.1-6)GalNAc-R 17
acetylglucosaminyltransferase peptide OGT 0- Y-S-D-S-P-S-T-S-T 18
.beta.-N-acetylglucosaminyltransferase Transferring
N-acetylglucosamine to an underlined sugar or amino acid.
TABLE-US-00002 TABLE 2 References 1 J Biol Chem. 10; 250(9): 3303-9
(1975) 2 Can J Biochem Cell Biol. 61(91): 1049-66. (1983) 3 J Biol
Chem. 10; 257(17): 10235-42. (1982) 4 J Biol Chem. 25; 258(10):
6162-73. (1983) 5 J Biol Chem. 25; 257(22): 13421-7. (1982) 6 J
Biol Chem. 20; 275(42): 32598-602. (2000) 7 Cell 105: 957-69 (2001)
8 J. Biol. Chem. 276 (5), 3498-507 (2001) 9 J Biol Chem. 276:
22032-40. (2001) 10 J Biol Chem. 12; 277(15): 12802-9. (2002) 11
Biochem. Biophys. Res. Commun. 294 (4), 843-8 (2002) 12 Nature 406:
411-5 (2000) 13 J. Biol. Chem. 259: 13385-90 (1984) 14 J. Biol.
Chem. 255: 11253-61 (1980) 15 J. Biol. Chem. 274: 3215-21 (1999) 16
J. Biol. Chem. 275: 11106-13 (2000) 17 Proc. Natl. Acad. Sci.
U.S.A. 96, 8991-6 (1999) 18 J. Biol. Chem. 265: 2563-2568
(1990)
REFERENCES
[0006] Reference 30: Kobata A., Eur. J. Biochem. 15, 209(2),
483-501, 1992 [0007] Reference 31: Santer U. V. et al., Cancer
Res., September, 44(9), 3730-5, 1984 [0008] Reference 32: Taniguchi
N., Biochim. Biophys. Acta., 1455(2-3), 287-300, 1999
[0009] As described above, the identification of gene products
having some involvement in canceration is expected to provide tumor
markers useful in cancer diagnosis. If especially a nucleic acid
found in a transcript can be used as an indicator for testing
canceration, only the identification of a transcript of a
particular gene can sufficiently provide an indicator useful in
testing canceration, without the need for elucidating the function
of its end product, for example, a protein. Especially the
identification of a nucleic acid has advantages which are not found
in immunoassay, because it can be performed on a DNA microarray and
a nucleic acid even in small amounts can also be quantified after
being amplified by PCR.
[0010] On the other hand, the function of oligosaccharides in
living bodies receives attention. However, the analysis of
oligosaccharide synthesis in living bodies does not necessarily
progress satisfactorily. This is partly because the mechanism of
oligosaccharide synthesis and the localization of sugar synthesis
in living bodies are not sufficiently elucidated. The analysis of
the mechanism of oligosaccharide synthesis requires the analysis of
enzymes synthesizing oligosaccharides, especially
glycosyltransferases, and analyzing which type of oligosaccharide
is generated with the enzyme. Therefore, there also has been a
growing demand for finding a novel glycosyltransferase and
analyzing its function.
DISCLOSURE OF THE INVENTION
[0011] In light of the above-described problems, an object of the
present invention is to provide a tumor marker nucleic acid
significantly altered in its transcription level following
canceration, a nucleic acid for testing canceration that targets
the tumor marker nucleic acid, and a method of testing canceration
using any of these nucleic acids.
[0012] Another object of the present invention is to provide a
nucleic acid encoding a novel human glycosyltransferase protein,
and the novel glycosyltransferase protein in the analysis of a
particular gene that receives attention as an indicator of the
canceration. The glycosyltransferase protein of the present
invention is especially a
.beta.1,3-N-acetyl-D-glucosaminyltransferase protein having an
activity of transferring N-acetyl-D-glucosamine to an acceptor
substrate through .beta.1,3-linkage.
[0013] A further alternative object of the present invention is to
provide a transformant expressing the nucleic acid in a host cell
as well as a method of growing the transformant to isolate the
protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the result of quantitative real-time PCR
analysis of G9 transcripts in various human tissues. Calibration
curves for G9 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)
were obtained by the serial dilution of the respective plasmid
DNAs. The expression level of the G9 transcript was adjusted to the
GAPDH measured for equivalent cDNA. The data was shown as the
average .+-.S.D. of values obtained from three experiments.
[0015] FIG. 2 shows a buffer in a reaction solution and pH
dependence, and metal ion dependence, which are plotted for the
activity of a G9 polypeptide. In FIG. 2A, the effect of pH on the
activity was assayed with cacodylate (filled square) and HEPES
(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (filled
circle) buffers. In FIG. 2B, the effect of a divalent cation was
assayed with varying concentrations of MnCl.sub.2 (filled circle),
CaCl.sub.2 (filled square), MgCl.sub.2 (filled triangle),
ZnCl.sub.2 (open circle), NiSO.sub.4 (open square), and CdSO.sub.4
(open triangle).
[0016] FIG. 3 shows the result of activity measurement when
oligosaccharides pyridylaminated with 2-aminopyridine (N-glycans)
are used as acceptor substrates. ND represents "not detected".
[0017] FIG. 4 is an electrophoretic picture showing activity
measurement when an .alpha.1-acidic glycoprotein (orosomucoid),
ovalbumin, and ovomucoid are used as acceptor substrates. The
reaction mixture of an enzyme with the substrate is treated either
without glycopeptidase F (-) or with glycopeptidase F (+), followed
by separation by SDS-PAGE. The gel is either stained with CBB
(Coomassie Brilliant Blue) (upper picture) or subjected to
autoradiography (lower picture).
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present inventors attempted the isolation and
purification of a nucleic acid of interest that seems to have high
sequence homology based on the nucleotide sequence of a gene of an
enzyme similar in action to an enzyme of interest. Specifically, at
first, the present inventors conducted a BLAST search using the
sequence of a .beta.1,3-N-acetylglucosaminyltransferase, a
glycosyltransferase known in the art, as a query sequence and
consequently found a genome sequence (GenBank No. AC011462) as a
sequence having homology.
[0019] Further, the present inventors successfully cloned a gene
encoding the protein by PCR and determined its nucleotide sequence
(SEQ ID NO: 1) and predicted amino acid sequence (SEQ ID NO.: 2). A
gene having the nucleotide sequence of SEQ ID NO: 1 was designated
as a G9 gene, and a protein having the amino acid sequence of SEQ
ID NO: 2 was designated as a G9 protein. The present inventors have
thereby completed the present invention by finding out that a
protein encoded by the nucleic acid is a novel glycosyltransferase
and that the presence or absence of the expression of the nucleic
acid or its expression level in cancerous tissues differs from that
in normal tissues. The present inventors further allowed the
expression of the nucleic acid obtained in the present invention by
a genetic engineering technique to yield a recombinant protein. The
investigation of the activity of a protein of the present invention
has revealed that the protein is a
.beta.1,3-N-acetyl-D-glucosaminyltransferase protein having an
activity of transferring N-acetyl-D-glucosamine from a donor
substrate to an acceptor substrate through .beta.1,3-linkage.
[0020] The present invention relates to a nucleic acid hybridizing
under stringent conditions to a nucleotide sequence described in
SEQ ID NO: 1 or a complementary nucleotide sequence thereof.
[0021] Preferably, the nucleic acid of the present invention
consists of a nucleotide sequence having at least 15 contiguous
nucleotides in a nucleotide sequence described in SEQ ID NO: 1 or a
complementary nucleotide sequence thereof.
[0022] The nucleic acid of the present invention is typically a
probe or a primer. The nucleic acid of the present invention can
also be a tumor marker.
[0023] The present invention also relates to a method of testing
the canceration of a biological sample, comprising:
[0024] (a) using any of the above-described nucleic acids to
measure the transcription level of the nucleic acid in a biological
sample; and
[0025] (b) diagnosing the biological sample as being cancerous when
the transcription level of the nucleic acid in the biological
sample significantly exceeds that in a normal biological sample as
a control.
[0026] According to a preferred aspect of the testing method by the
present invention, the method of testing the canceration of a
biological sample comprises:
[0027] (a) using any of the above-described nucleic acids as a
labeled probe, which is in turn brought into contact with a
biological sample under stringent hybridization conditions to
measure the transcription level of the nucleic acid in the
biological sample based on a signal from the label of the
hybridized nucleic acid; and
[0028] (b) diagnosing the biological sample as being cancerous when
the transcription level of the nucleic acid in the biological
sample significantly exceeds that in a normal biological sample as
a control.
[0029] According to another preferred aspect of the testing method
by the present invention, the method of testing the canceration of
a biological sample comprises:
[0030] (a) using the above-described primer that is labeled to
subject a biological sample to nucleic acid amplification and
measuring the amount of a resulting nucleic acid amplification
product; and
[0031] (b) diagnosing the biological sample as being cancerous when
the amount of the nucleic acid amplification product significantly
exceeds that in a normal biological sample as a control.
[0032] According to a further aspect of the testing method by the
present invention, the effectiveness of treatment for cancer
therapy can be examined by use of the nucleic acid of the present
invention.
[0033] The method of examining the effectiveness of treatment for
cancer therapy by the present invention is a method comprising:
[0034] using any of the nucleic acids according to the present
invention to measure the transcription level of the nucleic acid in
a biological sample that has received treatment for cancer therapy
and comparing its measurement value with that before the treatment
or without the treatment, thereby determining whether the treatment
given to the biological sample is effective or not.
[0035] A preferred aspect of the method of examining the
effectiveness of treatment by the present invention encompasses a
method comprising: using the biological sample which has already
been cancerous, and determining that treatment for cancer therapy
given to the biological sample is effective if the transcription
level of the nucleic acid in the biological sample that has
received the treatment is significantly below that before the
treatment or without the treatment.
[0036] The biological sample to which the method of examining the
effectiveness of treatment can be applied includes an in vivo
biological sample from a non-human model animal as well as an in
vitro biological sample derived from a tissue, a cell, or the like
(including a human tissue or cell, or the like). Alternatively the
biological sample to which each of the above-described methods
according to the present invention can be applied is typically a
sample derived from the large intestine or peripheral blood.
[0037] In other aspects of the present invention, the nucleotide
sequence of SEQ ID NO: 1 has 31% homology to those of known genes a
human .beta.1,3GlcNAc transferase 2 and a .beta.1,3Gal transferase
6, and a conserved motif therein is close to that in a .beta.1,3Gal
transferase. The nucleotide sequence has 60% homology to that of a
murine .beta.1,3GlcNAc transferase 1. The predicted amino acid
sequence of SEQ ID NO: 2 has a hydrophobic transmembrane region
characteristic of a glycosyltransferase at its N terminus.
[0038] From these points of view, the nucleic acid sequence of SEQ
ID NO: 1 presumably encodes a novel human glycosyltransferase that
transfers an N-acetyl-D-glucosamine residue to synthesize an
oligosaccharide through .beta.1,3-linkage. In actuality, an enzyme
protein having a biological activity was isolated and purified
therefrom and a certain activity was confirmed (Examples 4 and
5).
[0039] Since a protein consisting of the amino acid sequence of SEQ
ID NO: 2 has the activity of the novel glycosyltransferase,
providing an amino acid sequence of this novel protein and a
nucleic acid encoding it would make a contribution toward
satisfying diverse needs for them in the art.
[0040] That is, the present invention also relates to a
glycosyltransferase protein that transfers an
N-acetyl-D-glucosamine residue from a sugar donor substrate to a
sugar acceptor substrate through .beta.1,3-linkage and relates to a
nucleic acid encoding the protein. The typical sugar donor
substrate is UDP-GlcNac, and at least a Gal.beta.1,4GlcNAc
carbohydrate residue is the acceptor substrate.
[0041] Thus, the present invention also relates to a
.beta.1,3-N-acetyl-D-glucosamintyltransferase protein having an
activity of transferring N-acetyl-D-glucosamine from donor
substrate to an acceptor substrate through .beta.1,3-linkage,
wherein ".beta." represents an anomer having a cis configuration,
of anomers of glycosidic linkage at position 1 of the sugar
ring.
[0042] Moreover, the glycosyltransferase protein of the present
invention includes a glycosyltransferase protein that has at least
one of the following properties (a) to (c):
[0043] (a) acceptor substrate specificity:
[0044] the glycosyltransferase protein has a significant
transferring activity for at least Bz-.beta.-lactoside and/or
Gal.beta.1-4GlcNAc groups,
[0045] wherein "Bz" represents a benzyl group, "Gal" represents a
galactose residue, "GlcNAc" represents an N-acetyl-D-glucosamine
residue, and ".beta." represents an anomer having a cis
configuration, of anomers of glycosidic linkage at position 1 of
the sugar ring;
[0046] (b) reaction pH:
[0047] the glycosyltransferase protein has a high activity at or
around neutral; or
[0048] (c) divalent ion requirement:
[0049] the activity is enhanced in the presence of at least
Mn.sup.2+ or CO.sup.2+.
[0050] The glycosyltransferase protein of the present invention
particularly includes a glycosyltransferase protein that has a
significant activity for an acceptor substrate having an N-linked
oligosaccharide with four Gal.beta.1-4GlcNAc groups.
[0051] In addition, an aspect of the glycosyltransferase protein of
the present invention includes a glycosyltransferase protein that
has any one sequence of the following (A) to (C):
[0052] (A) any one amino acid sequence of SEQ ID NO: 2, SEQ ID NO:
16, or SEQ ID NO: 17;
[0053] (B) an amino acid sequence comprising any one amino acid
sequence of SEQ ID NO: 2, SEQ ID NO: 16, or SEQ ID NO: 17 in which
one or several amino acid(s) is(are) substituted, deleted, or
inserted; or
[0054] (C) an amino acid sequence having at least 40% identity to
any one amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 16, or SEQ
ID NO: 17.
[0055] In an alternative aspect, the present invention also relates
to a nucleic acid encoding a
.beta.1,3-N-acetyl-D-glucosaminyltransferase protein according to
any of the above-described aspects.
[0056] An aspect of the nucleic acid encoding a glycosyltransferase
protein of the present invention encompasses a nucleic acid that
comprises a full-length nucleotide sequence described in SEQ ID NO:
1, nucleotide sequence from nucleotide Nos. 76 to 1194 therein, a
nucleotide sequence from nucleotide Nos. 97 to 1194 therein, or any
of complementary nucleotide sequences thereof. Such a nucleic acid
can be DNA.
[0057] In a further alternative aspect, the present invention also
relates to a vector comprising a nucleic acid encoding a
glycosyltransferase protein as described above as well as a
transformant comprising the vector. The present invention further
relates to a method of producing a
.beta.1,3-N-acetyl-D-glucosaminyltransferase protein, comprising:
growing the above-described transformant and expressing the
glycosyltransferase protein to collect the glycosyltransferase
protein from the transformant.
[0058] In a still alternative aspect, the present invention can
provide an antibody recognizing a
.beta.1,3-N-acetyl-D-glucosaminyltransferase protein according to
any of the above-described aspects.
[0059] The finding that the nucleic acid of the present invention
encodes a novel glycosyltransferase protein suggests that the
expression level of the glycosyltransferase protein in a cancer
tissue exceeds that in a normal tissue. Accordingly, it may be
possible to test the canceration of a biological sample by
detecting or quantifying the protein of the present invention that
is expressed in the biological sample to compare its result with
that of a normal biological sample as a control.
[0060] Thus, the present invention also relates to a method of
testing the canceration of a biological sample, comprising the
steps of:
[0061] (a) detecting or quantifying the novel glycosyltransferase
protein of the present invention in a biological sample; and
[0062] (b) diagnosing the biological sample as being cancerous when
the quantified value of the glycosyltransferase protein in the
biological sample significantly exceeds that of the
glycosyltransferase protein in a normal biological sample as a
control.
[0063] Here, the use of an antibody specifically recognizing a
glycosyltransferase protein is exemplified for detecting the novel
glycosyltransferase protein.
PREFERRED MODE OF CARRYING OUT THE INVENTION
[0064] Hereinafter, the present invention will be described in
detail in accordance with embodiments of the present invention.
(1) Nucleic Acid of the Present Invention Involved in
Canceration
[0065] The present inventors have found that the canceration of a
normal tissue, for example, a human large intestine tissue, without
the expression of a nucleic acid having a nucleotide sequence
described in SEQ ID NO: 1 is confirmed to cause the nucleic acid to
be expressed therein, and that a normal tissue, for example,
peripheral blood from a patient with colorectal cancer, in which a
nucleic acid having a nucleotide sequence described in SEQ ID NO: 1
is generally expressed has a significant increase in the expression
level of the nucleic acid as compared to that in a normal
individual.
[0066] Thus, a nucleic acid consisting of the nucleotide sequence
of SEQ ID NO: 1 or a complementary sequence thereof is worthy of
note as a tumor marker useful in examination for a transcript in a
biological sample. According to the present invention, a nucleic
acid capable of specifically hybridizing under stringent conditions
to this tumor marker nucleic acid is provided.
[0067] A primer or probe according to the present invention is
typically a natural DNA fragment derived from a nucleic acid having
a nucleotide sequence of SEQ ID NO: 1, a synthetic DNA fragment
designed to have a nucleotide sequence of SEQ ID NO: 1, or any of
complementary strands thereof.
[0068] Especially the tumor marker nucleic acid was detected via a
BLAST search and is transcribed as mRNA encoding a structural gene.
In general, its full-length ORF or a portion thereof can be present
in a sample. From this point of view, using the nucleic acid as a
targeting primer or probe, a desired target sequence can be
selected from across the ORF of the nucleotide sequence of SEQ ID
NO: 1. The primer or probe of the present invention can be a
partial sequence in the nucleotide sequence of SEQ ID NO: 1.
[0069] Using the primer or probe as described above, the target
nucleic acid in a biological sample can be detected and/or
quantified as described below. Because a genomic sequence or the
like can be targeted, the nucleic acid of the present invention can
also be provided as an antisense primer for medical research or
gene therapy.
Probe of the Present Invention
[0070] When the nucleic acid of the present invention is used as a
probe, the nucleic acid is an oligonucleotide with 15 bases or
more, preferably 20 bases or more, selected from the nucleotide
sequence of SEQ ID NO: 1 or a complementary strand thereof, or
alternatively cDNA with a maximum length of a full-length ORF
region (i.e., 1191 bases: nucleotide Nos. 1 to 1191) in the
nucleotide sequence of SEQ ID NO: 1 or a complementary strand
thereof.
[0071] In particular, the probe of the present invention is widely
useful as a reagent or a diagnostic agent for medical research.
Considering that a nucleic acid having an exceedingly large
molecular weight is generally difficult to handle, a preferred base
length of the probe is exemplified by 50 to 500 bases, more
preferably 60 to 300 bases.
[0072] Depending on, for example, the base length or hybridization
conditions adopted, an oligonucleotide probe having a relatively
short strand can function as a probe even if there is a mismatch on
the order of one or several bases, especially one or two bases,
between the oligonucleotide probe and the nucleotide sequence of
SEQ ID NO: 1 or the complementary nucleotide sequence thereof. A
cDNA probe having a relatively long strand can function as a probe
even if there is a mismatch of 50% or less, preferably 20% or less,
between the cDNA probe and the nucleotide sequence of SEQ ID NO: 1
or the complementary nucleotide sequence thereof.
[0073] Alternatively, when the nucleic acid of the present
invention is a synthetic oligonucleotide, the number of bases
therein is 15 bases or more, preferably 20 bases or more. Depending
on the base length or hybridization conditions adopted, the
synthetic oligonucleotide can function as a probe even if there is
a mismatch on the order of one or several bases, especially one or
two bases, between the synthetic oligonucleotide and the nucleotide
sequence described in SEQ ID NO: 1 or the complementary nucleotide
sequence thereof.
[0074] It should be understood that the oligonucleotide probe
according to the present invention having 15 bases in length could
specifically hybridize under stringent conditions to the target
nucleic acid. Those skilled in the art can select a suitable
partial sequence with at least 15 bases from the nucleotide
sequence of SEQ ID NO: 1 according to various strategies concerning
oligonucleotide probe design known in the art. Moreover,
information from an amino acid sequence of SEQ ID NO: 2 would be
helpful in selecting a unique sequence likely to be suitable as a
probe.
[0075] "Under stringent conditions" used herein means hybridization
under moderately or highly stringent conditions. Specifically, the
moderately stringent conditions are based on, for example, the
length of DNA and can readily be determined by those having
ordinary skill in the art. Basic conditions are shown in Sambrook
et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Vol. 1,
7.42-7.45 Cold Spring Harbor Laboratory Press, 2001 and include,
for a nitrocellulose filter, the use of hybridization conditions
comprising a prewashing solution containing 5.times.SSC, 0.5% SDS,
1.0 mM EDTA (pH: 8.0) and a hybridization solution containing
approximately 50% formamide, 2.times.SSC to 6.times.SSC at
approximately 40 to 50.degree. C. (or other similar hybridization
solutions such as a Stark's solution in approximately 50% formamide
at approximately 42.degree. C.), and washing conditions comprising
0.5.times.SSC, 0.1% SDS at approximately 60.degree. C. The highly
stringent conditions are also based on, for example, the length of
DNA and can readily be determined by those skilled in the art. In
general, such conditions include hybridization and/or washing at a
temperature higher than that of the moderately stringent conditions
and/or a salt concentration lower than that of the moderately
stringent conditions and are defined by involving, for example, the
hybridization conditions as described above and washing in
0.2.times.SSC, 0.1% SDS at approximately 68.degree. C. Those
skilled in the art would appreciate that a temperature and a salt
concentration of a washing solution is optionally adjustable
according to factors such as the length of a probe.
[0076] As described above, those skilled in the art can readily
find and practice moderately or highly stringent conditions
suitable for a selected probe, based on common general technical
knowledge in various probe design methods and hybridization
conditions known in the art as well as empirical rules that would
be obtained through experimental means usually used.
[0077] The probe of the present invention includes a labeled probe
attached with a label such as fluorescent, radioactive, and biotin
labels, in order to detect or confirm the probe hybridized with the
target sequence. One example of the labeled probe according to the
present invention is an oligonucleotide consisting of a nucleotide
sequence of SEQ ID NO: 6 (which corresponds to a complementary
strand of a strand from nucleotide Nos. 485 to 502 in SEQ ID NO:
1). This labeled probe can be used for confirming or quantifying a
PCR product of the target nucleic acid. The labeled probe of the
present invention may also be integrated in a diagnostic DNA probe
kit or the like or may be immobilized on a chip such as a DNA
microarray.
Primer of the Present Invention
[0078] When the nucleic acid of the present invention is used as a
primer, the nucleic acid is an oligonucleotide. Specifically, two
regions are selected from the ORF region in the nucleotide sequence
of SEQ ID NO: 1 to satisfy the following conditions:
[0079] 1) each of the regions has a length of 15 bases or more,
preferably 18 bases or more, more preferably 21 bases or more and
no longer than 50 bases; and
[0080] 2) each of the regions has a G+C content of 40 to 70%.
Single-stranded DNAs having the same nucleotide sequences as those
of the two selected regions or nucleotide sequences complementary
to those of the regions may be produced, or otherwise the
single-stranded DNAs modified to maintain the binding specificity
for the nucleotide sequences may be produced. Preferably, the
primer of the present invention has a sequence completely
complementary to a partial sequence in the ORF region of SEQ ID NO:
1 and however, may have a one- or two-base mismatch.
[0081] One example of a pair of primers according to the present
invention is a pair of an oligonucleotide consisting of a
nucleotide sequence described in SEQ ID NO: 4 (which corresponds to
a complementary strand of a strand from nucleotide Nos. 450 to 469
in SEQ ID NO: 1) and an oligonucleotide consisting of a nucleotide
sequence described in SEQ ID NO: 5 (which corresponds to a
complementary strand of a strand from nucleotide Nos. 531 to 549 in
SEQ ID NO: 1).
[0082] A probe selected from the nucleotide sequence positioned
between a pair of primers used can be employed for quantifying the
target nucleic acid amplified by PCR. One example of a labeled
probe for detecting a PCR product is an oligonucleotide consisting
of a nucleotide sequence described in SEQ ID NO: 6 (which
corresponds to a complementary strand of a strand from nucleotide
Nos. 485 to 502 in SEQ ID NO: 1).
(2) Method of Testing Canceration According to the Present
Invention
[0083] According to a method of testing canceration of the present
invention, the transcription level of the target nucleic acid in a
transcript of a biological sample can be measured. Its measurement
result is compared with a result from a normal biological sample as
a control. If a significant difference lies between those results,
the biological sample can be diagnosed as being a cancerous
tissue.
[0084] In this testing method, a threshold normalized in advance on
the basis of known data concerning a normal biological sample may
be used as a detection result for a normal tissue used as a
control. For example, when a normal tissue is not obtained from an
identical patient as in the test of peripheral blood, comparison
with the average of values measured in normal individuals is
performed.
[0085] As used herein, the significant difference to be diagnosed
as being cancerous means that the substantial presence (i.e.,
significant concentration) of the target nucleic acid in a subject
tissue is confirmed if the target nucleic acid is expressed in a
normal tissue as in, for example, peripheral blood from a patient
with colorectal cancer, or that the concentration of the nucleic
acid in a subject tissue significantly exceeds that in a normal
tissue and preferably the nucleic acid in the subject tissue is not
less than 1.5 times, preferably 2 times, by concentration greater
than that in the normal tissue when the target nucleic acid is
generally expressed in a normal tissue as in, for example, a
colorectal cancer tissue.
[0086] The method of testing canceration according to the present
invention typically involves a hybridization assay and a PCR
method.
Hybridization Assay
[0087] Examples of a hybridization assay that can be used in the
present invention include various hybridization assays well known
to those skilled in the art such as a southern blot, northern blot,
dot blot, or a colony hybridization technique for a transcript
extracted from a biological sample.
[0088] Depending on the transcription amount of the target nucleic
acid or a difference from a normal tissue, a testing method known
in the art including a quantitative hybridization assay such as dot
blot or colony hybridization used alone or in combination with
immunoassay may be employed when the quantification of the target
nucleic acid or an increase in detection level is required.
[0089] According to the typical hybridization assay, a subject
nucleic acid extracted from a biological sample or an amplification
product thereof is immobilized on a solid phase and hybridized
under stringent conditions to a labeled probe to measure the label
bound to the solid phase after washing.
[0090] Every method known to those skilled in the art can be
applied to the extraction and purification of a transcript from a
biological sample. That purified from a biological sample and
subjected to the hybridization assay is typically cDNA from the
whole transcript of the biological sample. However, when
canceration is determined only by the substantial detection of the
target nucleic acid (i.e., when no target nucleic acid appears to
be expressed in a normal tissue), the use of a testing method such
as in situ hybridization in no need of the purification or the like
of a transcript would be practical for a subject tissue in clinical
tests.
Testing Method by Nucleic Acid Amplification
[0091] On the basis of "the nucleotide sequence of the nucleic acid
of the present invention", those skilled in the art can
appropriately create primers based on nucleotide sequences
positioned at both ends of the nucleic acid of the present
invention or a partial region of interest thereof to be prepared,
and readily amplify and prepare the region of interest by nucleic
acid amplification reaction (e.g., PCR) using the primers.
[0092] As used herein, examples of the nucleic acid amplification
reaction include reaction requiring thermal cycles such as
polymerase chain reaction (PCR) [Saiki R. K., et al., Science, 230,
1350-1354 (1985)], ligase chain reaction (LCR) [Wu D. Y., et al.,
Genomics, 4, 560-569 (1989); Barringer K. J., et al., Gene, 89,
117-122 (1990); Barany F., Proc. Natl. Acad. Sci. USA, 88, 189-193
(1991)], and transcription-based amplification [Kwoh D. Y., et al.,
Proc. Natl. Acad. Sci. USA, 86, 1173-1177 (1989)], and isothermal
reaction such as strand displacement amplification (SDA) [Walker G.
T., et al., Proc. Natl. Acad. Sci. USA, 89, 392-396 (1992); Walker
G. T., et al., Nuc. Acids Res., 20, 1691-1696 (1992)],
self-sustained sequence replication (3SR) [Guatelli J. C., Proc.
Natl. Acad. Sci. USA, 87, 1874-1878 (1990)], and Q.beta. replicase
system [Lizardi et al., BioTechnology 6, p. 1197-1202 (1988)]. For
example, Nucleic Acid Sequence-Based Amplification (NASBA) reaction
described in European Patent No. 0525882, which employs the
competitive amplification of a target nucleic acid with a mutant
sequence is also available. Preferably, the nucleic acid
amplification reaction is the PCR method.
[0093] The target nucleic acid in a transcript can be detected
using PCR method with, for example, a pair of primers of the
present invention selected from the target nucleic acid. In
general, the nucleic acid amplification method in itself, such as
PCR, is well known in the art and is readily carried out because a
reagent kit and an apparatus for the nucleic acid amplification
method are commercially available.
[0094] When the primer pair of the present invention is used to
carry out a nucleic acid amplification method by PCR with the
subject nucleic acid as a template, the subject nucleic acid
present in a sample is amplified while no amplification takes place
in a sample without the subject nucleic acid. Therefore, whether or
not the subject nucleic acid is present in the sample can be
determined by confirming the presence of an amplification product,
and the transcription level of the subject nucleic acid, that is,
the concentration thereof can also be determined by quantifying an
amplification product. PCR cycles when repeated a predetermined
number of times would amplify the subject nucleic acid to a desired
concentration. The nucleic acid in a normal tissue can also be
measured in a similar way. A nucleic acid of a gene extensively and
generally present in an identical tissue or the like, for example,
a nucleic acid encoding glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) or .beta.-actin may be used as a control.
[0095] The subject nucleic acid may be the whole mRNA as a
transcript extracted from a biological sample such as a subject
tissue or cell or may be the whole cDNA reverse-transcribed from
the mRNA. When mRNA as the subject nucleic acid is amplified, a
NASBA method (3SR method, TMA method) using the above-described
pair of primers may be adopted. The NASBA method in itself is well
known and can readily be practiced using the pair of primers
because a kit for the NASBA method is commercially available.
[0096] An amplification product obtained by PCR method can be
detected or quantified by procedures where the reaction solution
after amplification is subjected to electrophoresis and the
resulting bands are stained with ethidium bromide or the like or
procedures where the amplification product after electrophoresis is
immobilized on a solid phase such as a nylon membrane and
hybridized to a labeled probe specifically hybridizing to the
subject nucleic acid, for example, a labeled probe described in SEQ
ID NO: 6, to detect the label after washing.
[0097] When the "concentrations" of the target nucleic acids are
compared between a subject tissue and a normal tissue, it is
preferable to use a quantitative PCR method including a RT-PCR or
quantitative real-time PCR method for kinetic analysis. Because the
target nucleic acid previously purified is mRNA, a quantitative
real-time RT-PCR method is particularly preferred. However, the
quantification of the nucleic acid in the present testing method is
not limited to the quantitative PCR method, and other DNA
quantification methods known in the art such as northern blot, dot
blot, and a DNA microarray using the above-described probe for a
PCR product are applicable.
[0098] It is also possible to quantify the amount of the target
nucleic acid in a sample by carrying out quantitative RT-PCR using
quencher and reporter fluorescent dyes. Because especially a kit
for the quantitative RT-PCR is commercially available, the
quantitative RT-PCR can readily be carried out. In addition, it is
also possible to semi-quantify the target nucleic acid based on the
strength of electrophoretic bands.
Testing Method for Effectiveness of Cancer Therapy
[0099] The above-described method of testing canceration according
to the present invention can also be employed as a method of
examining the effectiveness of cancer therapy. Subjects to be
examined are the effectiveness of treatment for which the
effectiveness of cancer cure should be examined as well as the
effectiveness of treatment given to a cancerous cell or tissue or a
tumor tissue or the like obtained from a model animal for
experimental carcinogenesis. Such treatment includes every
prescription such as radiotherapy in addition to the administration
of an anticancer agent. The treatment is given to a cancerous
biological sample or to the focus of an experimental model
animal.
[0100] According to a method of examining the effectiveness of
cancer therapy according to the present invention, the
transcription level of the target nucleic acid in a biological
sample that has received treatment of interest is compared with
that before the treatment or without the treatment. Alternatively,
the transcription level may also be followed up after the
treatment. The treatment can be assessed as being effective for
cancer therapy if by using it the treatment causes the
transcription level to be significantly reduced or causes an
intentional rise in the transcription level to be significantly
suppressed.
[0101] Such examination includes determination about whether a
candidate substance of an anticancer agent given to a cancerous
tissue is effective or not, especially whether the candidate
substance is effective or not for a lesion tissue or the like in an
experimental model animal, and determination about whether a novel
candidate anticancer agent is effective or not for a patient with
cancer. To the contrary, assessment of whether carcinogenicity is
suppressed or not in an experimental model animal designed to
develop cancer, that is, whether an expected rise in the
transcription level is significantly suppressed or not is also
targeted.
[0102] In the present specification, the "transcription level" or
"transcription amount" of a nucleic acid refers to the abundance of
the nucleic acid derived from a transcript in a fixed amount of a
biological sample. Because a nucleic acid can be amplified for
quantification or the signal level of its label can be amplified,
the amount of the nucleic acid measured can also be expressed as an
amplified amount or an amplified signal level.
[0103] In the present specification, a "subject nucleic acid" or
"target nucleic acid" includes not only mRNA and siRNA but also
every type of nucleic acid obtained with mRNA as a template,
regardless of in vivo or in vitro origin.
[0104] In the present specification, a "biological sample" refers
to an organ, a tissue, and a cell as well as an experimental
animal-derived organ, tissue, and cell, etc., and is preferably a
tissue. The esophagus, the stomach, the pancreas, the liver, the
kidney, the duodenum, the small intestine, the large intestine, the
rectum, the colon, and peripheral blood are concretely exemplified.
Preferred are the large intestine, the rectum, the colon, and
peripheral blood, and more preferred are the large intestine and
peripheral blood. The term "measurement" used herein encompasses
any of detection, amplification, quantification, and
semi-quantification. The application of the nucleic acid of the
present invention also includes gene therapy.
[0105] The testing method of the present invention is a method of
testing the canceration of a biological sample as described above.
The phrase "testing canceration" used herein includes testing to
determine whether the biological sample develops cancer or not as
well as testing to determine whether malignancy is high or not, and
can be applied to diagnosis, therapy, and so on, for cancer in
medical care. The term "cancer" used herein typically refers to the
entire spectrum of malignant tumors and includes disease conditions
caused by a malignant tumor. The testing method of the present
invention is suitable for testing, but not limited to, cancer of
the esophagus, gastric cancer, cancer of the pancreas, cancer of
the liver, renal cancer, duodenal cancer, cancer of the small
intestine, colorectal cancer, cancer of the rectum, cancer of the
colon, and peripheral blood. Preferred are colorectal cancer,
cancer of the rectum, and cancer of the colon, and more preferred
is colorectal cancer.
(3) Nucleic Acid of the Present Invention Encoding Novel
Glycosyltransferase
[0106] Based on the finding of the nucleic acid described above,
the present invention also provides a nucleic acid encoding a
full-length novel glycosyltransferase protein or a fragment
thereof.
[0107] The nucleic acid of the present invention encoding a novel
glycosyltransferase is a nucleic acid consisting of a nucleotide
sequence described in SEQ ID NO: 1 or a complementary nucleotide
sequence thereof, preferably a nucleic acid consisting of a
nucleotide sequence from nucleotide Nos. 76 to 1194 in SEQ ID NO:
1. The nucleic acid of SEQ ID NO: 1 includes those encoding an
amino acid sequence of SEQ ID NO: 2, and the nucleic acid with the
nucleotide sequence from nucleotide Nos. 76 to 1194 in SEQ ID NO: 1
includes nucleic acids encoding amino acid sequences of SEQ ID NOs:
16 and 17. A nucleic acid encoding an amino acid sequence identical
to any amino acid sequence encoded by those nucleic acids because
of codon degeneracy is also encompassed by the present invention.
As previously described, these nucleic acids are nucleic acids
suitable for use in, for example, the method of testing
canceration.
[0108] The nucleic acid of the present invention encoding the novel
glycosyltransferase includes both single-stranded and
double-stranded DNAs and also includes RNA complements thereof.
Examples of the DNA include naturally-derived DNA recombinant DNA,
DNAs chemically bonded together, DNA amplified by PCR, and
combinations thereof. However, DNA is preferred in light of its
stability at the time of preparing a vector and a transformant.
[0109] The nucleic acid of the present invention may be prepared
by, for example, procedures below.
[0110] At first, a candidate gene likely to encode a homolog
protein of a .beta.1,3-N-acetylglucosaminyltransferase, a
glycosyltransferase known in the art, is searched, and its amino
acid (polypeptide) sequence is determined: when a program such as
BLAST is used to search a gene having a homology to a
.beta.1,3-N-acetylglucosaminyltransferase gene from a gene
database, for example, a human genome DNA sequence (AC011462: Homo
sapiens chromosome 19 clone CTC-435M10) and EST (expressed sequence
tag, AW444713) sequence likely to encode its homolog protein are
found.
[0111] A complementary sequence of the nucleic acid found as above
or a portion thereof is utilized to carry out nucleic acid
amplification reaction from a cDNA library or the like according to
a standard method using a basic genetic engineering approach such
as hybridization and nucleic acid amplification reaction, thereby
allowing the preparation of the nucleic acid of the present
invention. Because, for example, an approximately 1.2-kbp DNA
fragment is obtained as a PCR product, this fragment can be
separated by a method such as agarose gel electrophoresis, which
screens DNA fragments according to their molecular weights, and
then isolated according to a standard method such as a method for
cutting out a certain band.
[0112] Because its amino acid sequence is expected, from a
predicted amino acid sequence (SEQ ID NO: 2), to have a
transmembrane region at the N-terminus, the nucleic acid of the
present invention that encodes a solubilized form of a polypeptide
can also be obtained by preparing a region of a nucleotide sequence
encoding a polypeptide without the transmembrane region. In actual
experiments conducted by the present inventors, the removal of a
region from the N-terminus to the 26th to 33rd amino acid of the
amino acid sequence allowed the preparation of a polypeptide having
an enzyme activity of interest. Thus, a nucleic acid consisting of
a nucleotide sequence from nucleotide Nos. 76 to 1194 or from
nucleotide Nos. 97 to 1194 in SEQ ID NO: 1 is considered to contain
a region encoding an active domain region of the enzyme
protein.
[0113] A homologous nucleic acid cloned using the hybridization and
nucleic acid amplification reaction described above has at least
50% identity, preferably at least 60% identity, more preferably at
least 70% or more identity, even more preferably at least 80%
identity, still more preferably at least 90% or more identity, most
preferably at least 95% identity, to the nucleotide sequence
described in SEQ ID NO: 1. The nucleic acid of the present
invention is a nucleic acid encoding a
.beta.1,3-N-acetylglucosaminyltransferase protein. In addition to a
nucleic acid having the nucleotide sequence described in SEQ ID NO:
1, a nucleic acid encoding a protein (or a portion thereof) that is
similar in activity, function, property, or the like, also comes
within the scope of the present invention. The activity, property,
or the like, of a protein of the present invention is described
below in detail in Section (5) G9 enzyme protein of the present
invention. The nucleic acid of the present invention has the
closest identity to a .beta.1,3GlcNAc transferase 2 gene that is a
nucleic acid encoding a .beta.1,3-N-acetylglucosaminyltransferase
protein known in the art, and the identity of these two nucleic
acids is 31% in the total length and 51% in the active domain
(corresponding to SEQ ID NO: 17 of the present invention).
Accordingly, a nucleic acid that has, preferably at least 55%
identity to the nucleotide sequence described in SEQ ID NO: 1 and
encodes a .beta.1,3-N-acetylglucosaminyltransferase protein having
a similar property comes within the scope of the present
invention.
[0114] It is possible to determine the percentage of identity by
visual inspection and mathematical calculation. Or otherwise, the
percentage of identity of two nucleic acid sequences can be
determined by using the GAP computer program, version 6.0 described
in Devereux et al., Nucl. Acids Res. 12: 387, 1984 and available
from the University of Wisconsin Genetics Computer Group (UWGCG) to
compare sequence information. Preferred default parameters for the
GAP program include: (1) a unary comparison matrix (containing a
value of 1 for identity and 0 for non-identity) for nucleotide and
the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids
Res. 14: 6745, 1986 as described in Schwartz and Dayhoff, eds.,
Atlas of Protein Sequence and Structure, pp. 353-358, National
Biomedical Research Foundation, 1979; (2) a penalty of 3.0 for each
gap and an additional penalty of 0.10 for each symbol in each gap;
and (3) no penalty for end gaps. Other programs of sequence
comparison used by those skilled in the art are also available.
(4) Vectors and Transformants of the Present Invention
[0115] According to the present invention, a recombinant vector
containing the above-described nucleic acid that has been isolated
is provided. An example of a method for incorporating a DNA
fragment of the nucleic acid into a vector such as a plasmid
includes a method described in Sambrook, J. et al., Molecular
Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor
Laboratory, 1.1 (2001). Conveniently, a commercially-available
ligation kit (e.g., from TAKARA SHUZO) can also be used. The
recombinant vector (e.g., the recombinant plasmid) thus obtained is
introduced into a host cell (e.g., E. coli DH5.alpha., TB1, LE392,
or XL-LE392 or XL-1Blue).
[0116] A method for introducing a plasmid into a host cell includes
a calcium chloride method or calcium chloride/rubidium chloride
method, an electroporation method, an electroinjection method, a
method by chemical treatment such as PEG, and a method using a gene
gun or the like, described in Sambrook, J. et al., Molecular
Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor
Laboratory, 16.1 (2001).
[0117] The vector can be prepared simply by ligating a desired gene
with a vector for recombination (e.g., plasmid DNA) available in
the art according to a standard method. Concrete examples of the
vector used include, but are not limited to, pDONR201, pBluescript,
pUC18, pUC19, and pBR322 as a plasmid derived from E. coli.
[0118] Those skilled in the art can appropriately select
restriction ends to be compatible with an expression vector. Those
skilled in the art can appropriately select an expression vector
suitable for a host cell desired to express the enzyme of the
present invention. Thus, it is preferred that the expression vector
according to the present invention should be constructed so that
regions involved in gene expression (such as promoter, enhancer,
and operator regions) are properly arranged to allow the expression
of the nucleic acid in a host cell of interest and the nucleic acid
is properly expressed. The construction of the expression vector
can also employ a Gateway system (Invitrogen) which does not
require restriction treatment and ligation procedures. The Gateway
system is a system utilizing site-specific recombination, which
allows the cloning of a PCR product while maintaining its
orientation and also allows the subcloning of a DNA fragment into
an expression vector properly modified. Specifically, an entry
clone is created from a PCR product and a donor vector with a BP
clonase, a site-specific recombinase, and the PCR product is then
transferred to a destination vector that is capable of undergoing
recombination between the destination vector and the entry clone
through another recombinase LR clonase, thereby preparing an
expression clone compatible with an expression system. One of the
features of the Gateway system is that, once an entry clone is
initially created, no laborious subcloning step having procedures
with restriction enzymes and ligases is required.
[0119] The type of an expression vector is not particularly limited
as long as the expression vector has the function of expressing a
desired gene in a variety of prokaryotic and/or eukaryotic host
cells to produce a desired protein. Examples of a preferred
expression vector include: pQE-30, pQE-60, pMAL-C2, pMAL-p2, and
pSE420 for E. coli; pYES2 (Saccharomyces), and pPIC3.5K, pPIC9K,
and pA0815 (Pichia) for yeast; and pFastBac, pBacPAK8/9, pBK283,
pVL1392, and pBlueBac4.5 for insects.
[0120] The incorporation of the expression vector of the present
invention into a host cell can give a transformant. The host cell
may be a eukaryotic cell (such as a mammalian cell, yeast, and an
insect cell) or may be a prokaryotic cell (such as E. coli and B.
subtilis). A host cell for obtaining the transformant of the
present invention is not particularly limited and may also be a
cultured cell derived from humans (e.g., HeLa, 293T, and SH-SY5Y),
mice (e.g., Neuro2a, NIH3T3), and so on. Any of these are known in
the art and are commercially available (e.g., from DAINIPPON
PHARMACEUTICAL) or available from public research institutes (e.g.,
RIKEN Cell Bank). Alternatively, an embryo, an organ, a tissue, or
a non-human individual may also be used.
[0121] Incidentally, the nucleic acid of the present invention is a
nucleic acid found in a human genome library. Therefore, in the
present invention, by using a eukaryotic cell as a host cell for
the transformant of the present invention, the "nucleic acid of the
present invention" having a property close to that of a natural one
(e.g., morphology having the addition of an oligosaccharide) will
be obtained. Thus, it is preferable to select a eukaryotic cell,
especially a mammalian cell, as the "host cell". The mammalian cell
is concretely exemplified by a mouse-derived cell, and an animal
cell is exemplified by a mouse-derived, Xenopus laevis-derived,
rat-derived, hamster-derived, monkey-derived, or human-derived cell
or cultured cell lines established from those cells. E. coli,
yeast, or an insect cell used as a host cell is concretely
exemplified by DH5.alpha., M15, JM109, and BL21 (E. coli); INVScl
(Saccharomyces), and GS115 and KM 71 (Pichia) (yeast); and Sf21,
BmN4, and silkworm larva (insect cell).
[0122] When a bacterium, especially E. coli, is used as the host
cell, an expression vector is generally composed of at least a
promoter/operator region, an initiation codon, a gene encoding a
desired protein, a termination codon, a terminator, and a
replicable unit.
[0123] When yeast, a plant cell, an animal cell, or an insect cell
is used as a host cell, it is preferred that an expression vector
should generally contain at least a promoter, an initiation codon,
a gene encoding a desired protein, a termination codon, and a
terminator. For example, DNA encoding a signal peptide, an enhancer
sequence, 5'- and 3'-untranslated regions of a desired gene, a
selective marker region, or a replicable unit may optionally be
contained therein.
[0124] In the vector of the present invention, the preferred
initiation codon is exemplified by a methionine codon (ATG).
Moreover, the termination codon is exemplified by a termination
codon regularly used (e.g., TAG, TGA, and TAA).
[0125] The replicable unit means DNA having the ability to
replicate its total DNA sequence in a host cell and includes a
natural plasmid, an artificially-modified plasmid (plasmid prepared
from a natural plasmid), and a synthetic plasmid. The preferred
plasmid includes: a plasmid pQE30, pET, or pCAL, or
artificially-modified products thereof (DNA fragments obtained by
treating pQE30, pET, or pCAL with an appropriate restriction
enzyme) for E. coli; a plasmid pYES2 or pPIC9K for yeast; and a
plasmid pBacPAK8/9 for an insect cell.
[0126] Any of those usually used by those skilled in the art such
as enhancer and terminator sequences each derived from SV40 can be
used as enhancer and terminator sequences.
[0127] Any of those usually used can be used as a selective marker
according to a standard method. An example thereof, includes a gene
resistant to an antibiotic such as tetracycline, ampicillin, or
kanamycin or neomycin, hygromycin, or spectinomycin.
[0128] The expression vector can be prepared by consecutively and
circularly ligating at least the above-described promoter,
initiation codon, gene encoding a desired protein, termination
codon, and terminator region with an appropriate replicable unit.
On this occasion, an appropriate DNA fragment (e.g., a linker,
other restriction sites) can be used, if desired, according to a
standard method such as digestion with a restriction enzyme and
ligation using a T4 DNA ligase.
[0129] The introduction [transformation (transfection)] of the
expression vector of the present invention into a host cell can be
carried out using a method conventionally known in the art.
[0130] The expression vector can be transformed into, for example,
a bacterium (such as E. coli and Bacillus subtilis) by, for
example, a method of Cohen et al. [Proc. Natl. Acad. Sci. USA, 69,
2110 (1972)], a protoplast method [Mol. Gen. Genet., 168, 111
(1979)], and a competent method [J. Mol. Biol., 56, 209 (1971)],
into Saccharomyces cerevisiae by, for example, a method of Hinnen
et al. [Proc. Natl. Acad. Sci. USA, 75, 1927 (1978)] and a lithium
method [J. B. Bacteriol., 153, 163 (1983)], into a plant cell by,
for example, a leaf disk method [Science, 227, 129 (1985)] and an
electroporation method [Nature, 319, 791 (1986)], into an animal
cell by, for example, a method of Graham [Virology, 52, 456
(1973)], and into an insect cell by, for example, a method of
Summer et al. [Mol. Cell. Biol., 3, 2156-2165 (1983)],
respectively.
(5) Isolation and Purification of Enzyme Protein According to the
Present Invention
[0131] In recent years, an approach in which a transformant is
cultured and grown and a substance of interest is isolated and
purified from the cultured and grown products has been established
as a genetic engineering approach.
[0132] The enzyme protein according to the present invention can be
expressed (produced), for example, by culturing a transformant
containing the expression vector prepared as described above in a
nutrient medium. Preferably, the nutrient medium contains a carbon
source, an inorganic nitrogen source, or an organic nitrogen source
necessary for the growth of a host cell (transformant). The carbon
source is exemplified by glucose, dextran, soluble starch, sucrose,
and methanol. The inorganic nitrogen source or organic nitrogen
source is exemplified by ammonium salts, nitrates, an amino acid,
corn steep liquor, peptone, casein, a meat extract, soy bean cake,
and a potato extract. The nutrient medium may also contain other
nutrients (e.g., an inorganic salt (e.g., NaCl, calcium chloride,
sodium dihydrogenphosphate, and magnesium chloride), vitamins, an
antibiotic (e.g., tetracycline, neomycin, ampicillin, and
kanamycin)), if desired. Culture is carried out by a method known
in the art. Such culture conditions as temperature, pH of a medium,
and culture time are appropriately selected to produce the protein
according to the present invention in large amounts.
[0133] The protein according to the present invention can be
acquired from a cultured product obtained by the culture described
above, as follows: when the protein according to the present
invention is accumulated within a host cell, the host cell is
collected by a procedure such as centrifugation and filtration and
then suspended in an appropriated buffer (e.g., a buffer such as
Tris, phosphate, HEPES, and MES buffers having a concentration of
approximately 10 to 100 mM; pH is preferably in the range of 5.0 to
9.0, which differs depending on a buffer used), followed by the
disruption of the cell by a method suitable for the host cell used
to obtain contents of the host cell by centrifugation; whereas,
when the protein according to the present invention is secreted to
the outside of a host cell, the host cell is separated from a
medium by a procedure such as centrifugation and filtration to
obtain a culture filtrate. A host cell disruption solution or
culture filtrate can be subjected to the isolation and purification
of the protein either directly or after subjecting to ammonium
sulfate precipitation and dialysis. A method of isolating and
purifying the protein can include the following methods: a method
by affinity chromatography suitable for each tag generally used
when the protein is attached to a tag such as 6.times. histidine,
GST, or a maltose-binding protein; or alternatively a method that
will be described in detail in Examples below, that is, a method by
ion exchange chromatography, when the protein according to the
present invention is produced without such a tag. In addition, the
method can also include a method combining gel filtration,
hydrophobic chromatography, isoelectric chromatography, and so
on.
[0134] The enzyme protein according to the present invention is
allowed to act on a glycoprotein, oligosaccharide, or
polysaccharide, or the like, thereby transferring a certain sugar
residue. Thus, the enzyme according to the present invention can be
used in the modification of an oligosaccharide in a glycoprotein
and the synthesis of saccharides. In addition, by administering
this enzyme as an immunogen to an animal, an antigen against the
enzyme can be created, which can in turn be used to measure the
enzyme by immunoassay. Thus, the enzyme and the nucleic acid
encoding the enzyme according to the present invention are useful
for creating such an immunogen. Because an oligosaccharide
structure synthesized by this enzyme seems to be increased in a
cancer cell, an antibody against this oligosaccharide is probably
available as a tumor marker. Among antibodies against an
oligosaccharide, for example, CA19-9 is well known to be useful as
a tumor marker. Similarly, an oligosaccharide structure capable of
being a tumor antigen can be synthesized using the G9.
[0135] It is preferred that the expression vector of the present
invention should be constructed so that the enzyme is easily
isolated and purified as described above. When the enzyme is
prepared by a genetic engineering technique using the expression
vector according to the present invention that has been constructed
to be expressed especially in the morphology of a fusion protein
between a polypeptide having an enzyme activity and a labeling
peptide, its isolation and purification would easily be
performed.
[0136] An example of the identification (labeling) peptide
described above is a peptide having the function of allowing the
easy secretion/separation/purification or detection of the enzyme
according to the present invention from a grown product of a
transformant by expressing the enzyme according to the present
invention as a fusion protein between the identification peptide
and a polypeptide having an enzyme activity bound together in the
preparation of the enzyme by genetic recombination. Examples of
such an identification peptide include a peptide such as a signal
peptide (peptide consisting of 15 to 30 amino acid residues, which
is present in the N terminuses of many proteins and functions
within a cell for sorting out a protein in intracellular
transmembrane mechanisms: e.g., OmpA, OmpT, and Dsb), a protein
kinase A, a protein A (protein having a molecular weight of
approximately 42,000, which is a component of the cell wall of
Staphylococcus aureus), a glutathion S-transferase, a His tag
(sequence where 6 to 10 histidine residues are aligned and
arranged), a myc tag (13-amino acid sequence derived from a cMyc
protein), a FLAG peptide (analytical marker consisting of 8 amino
acid residues), a T7 tag (which consists of the first 11 amino acid
residues in a gene 10 protein), a S tag (which consists of 15 amino
acid residues derived from pancreatic RNase A), a HSV tag, pelB
(22-amino acid sequence in an E. coli outer membrane protein pelB),
a HA tag (which consists of 10 amino acid residues derived from
hemagglutinin), a Trx tag (thioredoxin sequence), a CBP tag
(calmodulin-binding peptide), a CBD tag (cellulose-binding domain),
a CBR tag (collagen-binding domain), .beta.-lac/blu (.beta.
lactamase), .beta.-gal (.beta. galactosidase), luc (luciferase),
HP-Thio (His-patch thioredoxin), HSP (heat shock peptide),
Ln.gamma. (laminin .gamma. peptide), Fn (partial peptide of
fibronectin), GFP (green fluorescent peptide), YFP (yellow
fluorescent peptide), CFP (cyan fluorescent peptide), BFP (blue
fluorescent peptide), DsRed and DsRed2 (red fluorescent peptides),
MBP (maltose-binding peptide), LacZ (lactose operator), IgG
(immunoglobulin G), avidin, and a protein G, and any of these
identification peptides may be used. Among them, especially the
signal peptide, protein kinase A, protein A, glutathione
S-transferase, His tag, myc tag, FLAG peptide, T7 tag, S tag, HSV
tag, pelB or HA tag is preferred because the enzyme according to
the present invention is expressed and purified more easily
according to a genetic engineering approach. It is particularly
preferable to obtain the enzyme according to the present invention
as a fusion protein with the FLAG peptide
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (SEQ ID NO: 6), because of
considerably excellent handling ease. The FLAG peptide is highly
antigenic, and it provides an epitope reversibly bound by a
specific monoclonal antibody and allows the rapid assay and easy
purification of a recombinant protein expressed. A murine hybridoma
designated as 4E11 produces a monoclonal antibody that binds to the
FLAG peptide in the presence of a certain divalent metal cation, as
described in U.S. Pat. No. 5,011,912 (incorporated herein by
reference). The 4E11 hybridoma cell line is deposited in American
Type Culture Collection under Accession No. HB 9259. The monoclonal
antibody binding to the FLAG peptide is available from Eastman
Kodak Co., Scientific Imaging Systems Division, New Haven,
Conn.
[0137] A basic vector capable of being expressed in a mammalian
cell and yielding the enzyme according to the present invention as
a fusion protein with the FLAG peptide is, for example, pFLAG-CMV-1
(manufactured by Sigma-Aldrich). Alternatively, a vector capable of
being expressed in an insect cell is exemplified by pFBIF (a vector
in which a region encoding the FLAG peptide is incorporated into
pFastBac (Invitrogen): see Examples below). However, those skilled
in the art can select a suitable basic vector, judging from a host
cell, a restriction enzyme, an identification peptide, and so on,
used in the expression of the enzyme.
(6) G9 Enzyme Protein of the Present Invention
[0138] As described above, a polypeptide having a certain enzyme
activity can be isolated and purified using the G9 nucleic acid of
SEQ ID NO: 1 of the present invention on the basis of a genetic
engineering approach.
[0139] First, from the above point of view, a typical aspect of the
protein of the present invention is a G9 enzyme protein having an
amino acid sequence of SEQ ID NO: 2 predicted from the nucleic acid
sequence of SEQ ID NO: 1. Specifically, this enzyme protein has
activities described below.
Catalytic Reaction
[0140] The G9 enzyme protein can transfer N-acetyl-D-glucosamine
(GlcNAc) from its donor substrate to its acceptor substrate through
.beta.1,3 glycosidic linkage and synthesize an oligosaccharide.
Donor Substrate Specificity:
[0141] Examples of the N-acetyl-D-glucosamine donor substrate
include a sugar nucleotide having this sugar residue, for example,
uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), adenosine
diphosphate-N-glucosamine (ADP-GlcNAc), guanosine
diphosphate-N-acetylglucosamine (GDP-GlcNAc), and cytidine
diphosphate-N-acetylglucosamine (CDP-GlcNAc). The typical donor
substrate is UDP-GlcNAc.
Acceptor Substrate Specificity (See Table 5):
[0142] The typical acceptor substrate exhibits a significant
activity for Bz-.beta.-lactoside and Gal.beta.1-4GlcNAc.alpha.-pNp
and a particularly strong activity for
Gal.beta.1-4GlcNAc.alpha.-pNp, of pNp-.alpha.-Glc, pNp-.beta.-Glc,
pNp-.alpha.-GlcNAc, pNp-.beta.-GlcNAc, pNp-.alpha.-Gal,
oNp-.beta.-Gal, pNp-.alpha.-GalNAc, pNp-.alpha.-Xyl,
oNp-.beta.-Xyl, pNp-.alpha.-Fuc, Bz-.alpha.-Man, Bz-.alpha.-ManNAc,
Bz-.beta.-lactoside, GlcNAc.beta.1-4GlcNAc.beta.-BZ,
Gal.beta.1-4GlcNAc.alpha.-pNp.
[0143] In the present specification, "GlcNAc" represents a
N-acetyl-D-glucosamine residue; "GalNAc" represents a
N-acetyl-D-galactosamine residue; "ManNAc" represents a
N-acetyl-D-mannosamine residue; "Glc" represents a glucose residue;
"Man" represents a mannose residue; "Gal" represents a galactose
residue; "Bz" represents a benzyl group; "pNp" represents a
paranitrophenyl group; "oNp" represents an orthonitrophenyl group;
and "-" represents glycosidic linkage. The number in the formula
represents a carbon number of glycosidic linkage present in the
oligosaccharide. Besides, ".alpha." and ".beta." represent anomers
of the glycosidic linkage at position 1 of the sugar ring, and the
anomer trans to CH.sub.2OH or CH.sub.3 at position 5 and the anomer
cis thereto in positional relationship are expressed by ".alpha."
and ".beta.", respectively.
[0144] Thus, the G9 enzyme protein of the present invention
catalyzes reaction given by, for example, the following
formula:
UDP-GlcNAc+Gal.beta.1-4GlcNAc.alpha.-R.fwdarw.UDP+GlcNAc.beta.1-3Gal.bet-
a.1-4GlcNac.alpha.-R,
wherein R is a glycoprotein, glycolipid, oligosaccharide, or
polysaccharide, or the like, having the GlcNAc residue.
[0145] The G9 enzyme protein of the present invention exhibits the
transferring activity for an oligosaccharide (e.g., oligosaccharide
pyridylaminated with 2-aminopyridine) or a glycoprotein having an
oligosaccharide residue. The G9 enzyme protein exhibits a
significantly strong activity or a selectively significant activity
for especially a substrate having the oligosaccharide residue in a
quadruple-stranded form at the nonreducing end of an N-linked
oligosaccharide (see FIGS. 3 and 4).
Optimum Buffer and Optimum pH (see FIG. 2A):
[0146] The G9 enzyme protein has the above-described catalysis in
both sodium cacodylate and HEPES buffers. Generally, it has a high
activity at or around neutral. Concerning the pH dependence of an
activity in each of the buffers, the G9 enzyme protein has an
activity that rises with an increase in pH within the neutral
region from pH 6.4 to 7.2 in the sodium cacodylate buffer and shows
the maximum activity around pH 7.0 in the HEPES buffer.
Divalent Ion Requirement (See FIG. 2B):
[0147] The activity of the human G9 protein is significantly
enhanced in the presence of at least Mn ion or Co ion among
divalent metal ions and particularly remarkably enhanced in the
presence of Mn ion. These enhanced activities rapidly rise in the
low concentration region of the ion and subsequently gradually
decrease. Although Cd and Ni ions show slight enhancing effect in
the low concentration region, no enhancing effect is substantially
observed in Mg and Zn ions. Usually, most of glycosyltransferases
requiring a divalent ion have reaction enhanced in the presence of
Mn ion.
[0148] As described above, the G9 enzyme protein of the present
invention can transfer a GlcNAc residue to a certain
oligosaccharide through .beta.1,3 glycosidic linkage under the
given enzyme reaction conditions described above and as such, is
useful in the synthesis or modification reaction of an
oligosaccharide of a glycoprotein or the like.
[0149] Second, in the present specification, the disclosure of the
amino acid sequence described in SEQ ID NO: 2 that represents the
primary structure of the above-described protein provides every
type of protein that can be produced by a genetic engineering
approach well known in the art on the basis of the amino acid
sequence (hereinafter, also referred to as a "mutant protein" or
"modified protein"). That is, the enzyme protein of the present
invention is not limited to a protein consisting of the predicted
amino acid sequence of SEQ ID NO: 2 according to common general
technical knowledge in the art and as illustrated below, is
intended to include a protein consisting of a polypeptide being not
full-length that partially lacks, for example, the N-terminal side
of the amino acid sequence, or even a protein with an amino acid
sequence homologous to those amino acid sequences, which has
intrinsic properties of the proteins.
[0150] Initially, the human G9 enzyme protein of the present
invention can be composed of preferably an amino acid sequence
described in SEQ ID NO: 16 (amino acid sequence from amino acid No.
26 to the C terminus in SEQ ID NO: 2), more preferably an amino
acid sequence described in SEQ ID NO: 17 (amino acid sequence from
amino acid No. 33 to the C terminus in SEQ ID NO: 2), as has been
obtained in the example below.
[0151] In general, it is well known that a protein having
physiological activities such as an enzyme can maintain the
physiological activities even if one or several amino acid(s)
is(are) substituted or deleted in any of the above-described amino
acid sequences or one or several amino acid(s) is(are) inserted or
added to any of the amino acid sequences. It is also known that
there exist mutant proteins among native proteins, which have the
mutation of one or several amino acid(s) due to the variety of
living species producing them, gene mutation caused by variations
in ecotype, or the presence of greatly similar isozymes, or the
like. From this point of view, the protein of the present invention
also includes a mutant protein that has an amino acid sequence
comprising each amino acid sequence shown in SEQ ID NO: 2, 16, or
17 in which one or several amino acid(s) is(are) substituted or
deleted or to which one or several amino acid(s) is(are) inserted
or added, and has an activity of transferring a GlcNAc residue to a
Gal residue through .beta.1,3-glycosidic linkage under a certain
enzyme reaction conditions described above. In addition, the
particularly preferred modified protein includes a protein that has
an amino acid sequence comprising each amino acid sequence shown in
SEQ ID NO: 2 in which one or several amino acid(s) is(are)
substituted or deleted or to which one or several amino acid(s)
is(are) inserted or added.
[0152] In the description above, "several" refers to preferably 1
to 200, more preferably 1 to 100, even more preferably 1 to 50,
most preferably 1 to 20. In general, the numbers of amino acids
that can be substituted with the activity of the original protein
maintained when an amino acid is substituted by site-specific
mutation is preferably 1 to 10.
[0153] Moreover, the modified protein of the present invention
includes a modified protein obtained by the substitution of an
amino acid by another having a property similar thereto. That is,
an approach for creating a recombinant protein having desired
mutation by introducing the substitution of an amino acid by
another having a property similar thereto (e.g., the substitution
of a hydrophobic amino acid by another hydrophobic amino acid, the
substitution of a hydrophilic amino acid by another hydrophilic
amino acid, the substitution of an acidic amino acid by another
acidic amino acid, or the substitution of a basic amino acid by
another basic amino acid) is well known to those skilled in the
art, and most of the modified proteins thus obtained have
properties similar to those of the original proteins. From this
point of view, a modified protein allowed to have such amino acid
substitution is also encompassed by the present invention.
[0154] In addition, the modified protein of the present invention
may be a glycoprotein in which an oligosaccharide binds to the
polypeptide as long as it has the amino acid sequence as described
above and has an enzyme activity intrinsic to an enzyme of
interest.
[0155] When the amino acid sequence of the present invention is
subjected to an identity search provided by GENETYX for identifying
the range of the homologous protein of the present invention, it is
understood that the amino acid sequence has the highest identity to
that of a .beta.1,3-N-acetylglucosaminyl transferase 2 protein
known in the art, and that the identity of these two amino acid
sequences is 37% in the total length and 39% in the active domain
(which corresponds to SEQ ID NO: 17). From this point of view, an
amino acid sequence preferred as the homologous protein of the
present invention may generally have at least 40% identity, more
preferably at least 50% identity, particularly preferably at least
60% identity to the amino acid sequence shown in SEQ ID NO: 2, 16,
or 17. The .beta.1,3-N-acetylglucosaminyltransferase protein having
a property similar to those of proteins having the amino acid
sequences comes within the scope of the present invention.
[0156] The GENETYX is genetic information processing software for
nucleic acid analysis and protein analysis and is capable of signal
peptide prediction, promoter site prediction, and secondary
structure prediction in addition to usual homology analysis and
multi-alignment analysis. The homology analysis program used herein
adopts Lipman-Pearson method (Lipman, D. J. & Pearson, W. R.,
Science, 277, 1435-1441 (1985)) that is frequently used as a method
with high throughput and high sensitivity.
[0157] In the present specification, percentage of identity may be
determined by the comparison with sequence information using, for
example, a BLAST program described by Altschul et al. (Nucl. Acids.
Res., 25. 3389-3402 (1997)) or FASTA described by Pearson et al.
(Proc. Natl. Acad. Sci. USA, 2444-2448 (1988)). The programs are
available on the Internet from the website of National Center for
Biotechnology Information (NCBI) or DNA Data Bank of Japan (DDBJ).
A variety of conditions (parameters) for an identity search
provided by each of the programs are described in detail in the
website. Although a part of the configurations can appropriately be
altered, the search is generally conducted using default values. It
is noted that other sequence comparison programs used by those
skilled in the art may also be employed.
[0158] Third, as described below, the isolated protein of the
present invention can be administered as an immunogen to an animal
to thereby produce an antibody against the protein. The enzyme can
be measured and quantified by immunoassay using such an antibody.
Thus, the present invention is useful for creating such an
immunogen. From this point of view, the protein of the present
invention includes a polypeptide fragment, a mutant, and a fusion
protein from the protein, which contains an antigenic determinant
or epitope for eliciting the formation of an antibody.
(7) Antibody Recognizing Protein According to the Present
Invention
[0159] In the present specification, an antibody immunoreactive to
a glycosyltransferase protein encoded by the nucleic acid of the
present invention is provided. Such an antibody specifically binds
to the glycosyltransferase protein via the antigen-binding site of
the antibody (in contrast to non-specific binding). Thus, the
protein of SEQ ID NO: 2, 16, or 17, or a fragment, mutant, or
fusion protein thereof, or the like, as described above may be used
as an "immunogen" for producing an antibody immunoreactive thereto.
To be more specific, the protein, the fragment, mutant, and fusion
protein thereof, and so on, contain an antigenic determinant or
epitope for eliciting the formation of an antibody. The antigenic
determinant or epitope may be either linear or conformational
(interrupted). The antigenic determinant or epitope may be
identified by any method known in the art.
[0160] Thus, one aspect of the present invention relates to an
antigenic epitope of a glycosyltransferase protein encoded by the
nucleic acid of the present invention. Such an epitope is useful
for creating an antibody, especially a monoclonal antibody, as more
fully described below. Furthermore, the epitope of the
glycosyltransferase protein according to the present invention may
be used in assay and as a research reagent for purifying an
antibody specifically bound with the epitope from a substance such
as a supernatant derived from polyclonal serum or a cultured
hybridoma. Such an epitope or a mutant thereof can be produced
using a technique known in the art such as solid phase synthesis
and chemical or enzymatic cleavage of a protein, or using a
recombinant DNA technique.
[0161] For an antibody likely to be induced by the
glycosyltransferase protein, both polyclonal and monoclonal
antibodies can be prepared by a routine technique even if the
whole-protein or a portion thereof is isolated or the epitope is
isolated. See, e.g., Kennet et al., ed., Monoclonal Antibodies,
Hybridomas: A New Dimension in Biological Analyses, Plenum Press,
New York, 1980.
[0162] The present specification is also directed to a hybridoma
cell line that produces a monoclonal antibody specific to the
glycosyltransferase protein of the present invention. Such a
hybridoma can be produced and identified by a routine technique.
One method for producing such a hybridoma cell line comprises:
immunizing an animal with the glycosyltransferase protein;
collecting spleen cells from the immunized animal; fusing the
spleen cells with myeloma cell lines to thereby produce hybridoma
cells; and identifying a hybridoma cell line producing a monoclonal
antibody that binds to the enzyme. The monoclonal antibody can be
collected by a routine technique.
[0163] The monoclonal antibody according to the present invention
includes a chimeric antibody, for example, a humanized murine
monoclonal antibody. When such a humanized antibody is prepared by
a known technique and the antibody is administered to a human, an
advantage of reduced immunogenicity may be provided.
[0164] An antigen-binding fragment of an antibody, which can be
produced by a routine technique, is also encompassed by the present
invention. Examples of such a fragment include, but are not limited
to, Fab and F(ab').sub.2. An antibody fragment and a derivative
produced by a genetic engineering technique are also provided.
[0165] The antibody according to the present invention can be used
in assay for detecting the presence of the glycosyltransferase
protein or a fragment thereof either in vitro or in vivo. The
antibody can also be employed when the polypeptide of the present
invention or a fragment thereof is purified by immunoaffinity
chromatography.
[0166] Furthermore, the use of a binding partner, for example, an
antibody capable of blocking the binding of the glycosyltransferase
protein to an acceptor substrate allows the inhibition of a
biological activity generated from such binding. Such a blocking
antibody may be identified using any suitable assay method such as
the test of antibody for the ability to inhibit the binding of the
protein to a certain cell in which an acceptor substrate is being
expressed. Alternatively, the blocking antibody can be identified
in assay for the ability to inhibit biological influence arising
from the enzyme according to the present invention bound with a
binding partner for a target cell.
[0167] By using such an antibody an in vitro method or
administering the antibody in vivo, a biological activity mediated
by an entity producing the antibody can be inhibited. Thus, it is
possible to treat disorders caused or deteriorated (directly or
indirectly) by the interaction between the glycosyltransferase
protein of the present invention and a binding partner. The therapy
involves administrating, to a mammal in vivo, the blocking antibody
in an effective amount for inhibiting a binding partner-mediated
biological activity. In general, a monoclonal antibody is preferred
for use in such therapy. In one aspect, an antigen-binding antibody
fragment is used.
(8) Finding of Murine G9 and Construction of Genetically-Engineered
Animal
[0168] As the above-described human G9 has been found, the present
inventors have conducted a search from a gene database using a
BLAST search and so on and consequently detected the presence of
murine orthologs of the human G9 (SEQ ID NOs: 18 to 20).
[0169] A murine G9 nucleic acid has been found as a nucleic acid
having ORF with 1170 bases in length (SEQ ID NO: 19) in a sequence
with a total of 1845 bases in length (SEQ ID NO: 18). This ORF is
estimated to encode an amino acid sequence (SEQ ID NO: 20) of a
murine .beta.1,3-N-acetyl-D-glucosaminyltransferase protein
consisting of 389 amino acid residues. In the present
specification, every investigation and deliberation conducted on
the human G9 can similarly be applied to the murine G9.
[0170] Based on the finding of the murine G9, the present invention
also provides means for the expression and function analysis of the
G9 at the animal individual level which is based on a variety of
gene conversion techniques using fertilized eggs and ES cells and
typically provides even a transgenic animal into which the G9 gene
is introduced and a knockout mouse that is deficient in the murine
G9.
[0171] For example, the knockout mouse can be constructed according
to a standard method in the art (see e.g., The Latest Techniques
for Gene Targeting, K. Yagi, ed., YODOSHA; Gene Targeting,
supervisory for translation by T. Noda, MEDICAL SCIENCES
INTERNATIONAL). That is, those skilled in the art can acquire a
homologously recombinant ES cell of the murine G9 (mG9) according
to a gene targeting method known in the art using the sequence
information of murine G9 nucleic acid disclosed in the present
application and can construct a G9 knockout mouse using the ES cell
(see Example 6).
[0172] Alternatively, a method for suppressing gene expression by a
small interfering RNA method has recently been developed (T. R.
Brummelkamp et al., Science, 296, 550-553 (2002)), and the G9
knockout mouse can be constructed according to such a method known
in the art.
[0173] Providing the G9 knockout mouse would be helpful in
elucidating the involvement of the G9 gene in a certain life
phenomenon, that is, information on the redundancy of the gene as
well as the relationship between the deficiency of the gene and a
phenotype (including every type of abnormality for motion,
intelligence, and sensory function) at an individual level and even
the function of the gene in individual lifecycle such as
development, growth, and aging. To be more specific, the knockout
mouse obtained by the above-described method can be used to
investigate the detection of the carriers of oligosaccharides
synthesized by the G9 and mG9 and the relationship with
physiological function and disease, and so on. For example, a
glycoprotein and a glycolipid are extracted from each tissue
excised from the knockout mouse and compared with those from a
wild-type mouse by a technique such as proteomics (e.g.,
two-dimensional electrophoresis, two-dimensional thin-layer
chromatography, and mass spectrometry) to thereby allow the
identification of the carriers of the synthesized oligosaccharides.
Moreover, the comparison of phenotypes (e.g., fetation, growth
process, and spontaneous behavior) between the knockout mouse and a
wild-type mouse allows the estimation of the relationship with
physiological function and disease.
[0174] Hereinafter, the present invention will be described more
fully with reference to examples. However, the present invention is
not intended to be limited to examples described below.
EXAMPLES
Example 1 Cloning of DNA of the Present Invention
[0175] As a result of searching a gene having homology to a
.beta.1,3-N-acetylglucosaminyltransferase gene or a gene likely to
encode a protein having homology to the enzyme at an amino acid
level from a gene database using programs such as Blast [Altschul
et al., J. Mol. Biol. 215, 403-410 (1990)], the FASTA method, the
PSI-BLAST method, and the FrameSearch method [manufactured by
Compugen], a human genome DNA sequence (AC011462: Homo sapiens
chromosome 19 clone CTC-435M10) and a EST (AW444713) sequence, and
the like were found. A polypeptide encoded by each of these
sequences is considered to be a homolog protein of the
.beta.1,3-N-acetylglucosaminyltransferase and was designated as
G9.
[0176] Unless otherwise specified in the description below, a
method known in the art which is described in Molecular Cloning 2nd
Ed. was used as a genetic engineering approach.
[0177] RNA was extracted from a colorectal cancer cell line colo
205 with RNeasy Mini Kit (manufactured by Qiagen) to synthesize
single strand DNA by an oligo(dT) method using Super-Script
First-Strand Synthesis System (manufactured by Invitrogen). This
DNA was used as a template to carry out PCR with a 5' primer (SEQ
ID NO: 3) and a 3' primer (SEQ ID NO: 4). PCR conditions comprised
25 cycles, each cycle having 94.degree. C. for 30 seconds,
65.degree. C. for 1 minute, and 72.degree. C. for 1 minute. A DNA
fragment obtained by PCR has, as restriction sites, HindIII on the
5' side of the initiation codon of ORF and EcoRI on the 3' side of
the stop codon thereof.
[0178] This DNA fragment and pBluescript(R) II SK(-) (manufactured
by TOYOBO) were individually treated with restriction enzymes
HindIII and EcoRI, with which a reaction solution was then mixed,
followed by ligation reaction to thereby introduce the ORF of the
G9 into the pBluescript(R) II SK(-). The reaction solution was
purified by an ethanol precipitation method and then mixed with a
competent cell (E. coli DH5.alpha.). The mixture was subjected to a
heat shock method (42.degree. C., 30 sec.) and seeded to a LB agar
medium containing IPTG and X-gal. The next day, a single white
colony was further cultured to collect plasmid DNA.
[0179] The collected plasmid DNA was confirmed to contain the
nucleic acid sequence of interest and the nucleotide sequence was
determined (SEQ ID NO: 1). A predicted open reading frame (ORF) in
that nucleotide sequence is 1194 bp, and its predicted amino acid
sequence consists of 397 amino acids (SEQ ID NO: 2). The predicted
amino acid sequence has a hydrophobic amino acid region
characteristic of a glycosyltransferase at its N terminus. Those
consisting of the nucleic acid sequence and the amino acid sequence
were designated as G9.
[0180] The pBluescript(R) II SK (-) into which the G9 is
incorporated is a multifunctional vector developed for carrying out
cloning procedures and sequencing procedures more conveniently and
has various improvements in addition to the function of
conventional pUC and M13 vectors. Because a multi-cloning site is
contained in a LacZ gene as with the pUC vector, the plasmid
incorporating an insert therein is transformed into E. coli having
the genotype of lacZ.DELTA.M15 such as XL1-Blue MRF' and JM109,
which in turn forms a white colony in a plate supplemented with
IPTG/X-gal. Thus, the presence or absence of the insert can be
easily assessed. Moreover, because the multi-cloning site has a
polylinker consisting of 21 restriction sites, the range of choices
for restriction enzymes used are extended when a deletion mutant is
created by Exo/Mung System. The incorporated G9 gene can be
adjusted in its expression within E. coli having lacIq mutation
through LacZ operator/promoter, and the E. coli is allowed to
produce a protein of interest by the addition of IPTG to a medium.
Furthermore, because T3 and T7 promoters are present on both sides
of that multi-cloning site, it is possible to create RNA probes
with these promoters. BssHII sites are present on both sides of
each of those promoters and can be utilized to cut out inserted DNA
together with the promoter sequences. Using the probes from both of
the promoters, gene mapping can be carried out. Because such a
vector contains the replication origin of an f1 phage,
single-stranded DNA is produced by the infection of a VCSM13 or
R408 helper phage and can be used in sequencing and Site Specific
Mutagenesis. An antisense strand is rescued by the infection of the
helper phage.
Example 2 Expression Level of DNA of the Present Invention in Human
Colorectal Cancer Tissue
[0181] The expression level of the G9 gene in normal and colorectal
cancer tissues from identical patients were compared using a
quantitative real-time PCR method.
[0182] The quantitative real-time PCR method is a method that
combines sense and antisense primers with a fluorescently-labeled
probe in PCR. In amplification by PCR, the fluorescent label of the
probe comes off and shows fluorescence. Fluorescent intensity is
amplified in correlation with the amplification of a gene and as
such, used as an indicator to conduct quantification.
[0183] RNA was extracted from human colorectal cancer tissues and
normal and colorectal cancer tissues from identical patients with
RNeasy Mini Kit (manufactured by Qiagen) to synthesize single
strand DNA by an oligo(dT) method using Super-Script First-Strand
Synthesis System (manufactured by Invitrogen). This DNA was used as
a template to carry out quantitative real-time PCR with ABI PRISM
7700 (manufactured by Applied Biosystems Japan) using a 5' primer
(SEQ ID NO: 5), a 3' primer (SEQ ID NO: 6), and a TaqMan probe (SEQ
ID NO: 7). PCR conditions comprised reaction at 50.degree. C. for 2
minutes and 95.degree. C. for 10 minutes, followed by cycles
repeated 50 times, each cycle having 95.degree. C. for 15 seconds
and 60.degree. C. for 1 minute. The obtained measurement values
were divided by a value from .beta.-actin as an internal standard
gene quantified using a kit manufactured by Applied Biosystems
Japan Ltd., in order to correct variations among individuals.
Comparison was made between the measurement values of the human
colorectal cancer tissues and those of the normal and colorectal
cancer tissues from identical patients.
[0184] The result has demonstrated that the transcript from the DNA
of the present invention is not present or is too negligible to
measure in the non-cancerous tissues and that the transcript from
the DNA of the present invention is significantly present in the
cancerous tissues (see Table 3).
TABLE-US-00003 TABLE 3 Patient Normal Cancer No. tissue tissue
Patient 1 0 0.0052 Patient 2 0 0.0004 Patient 3 0 0.0023 Patient 4
0 0.0012 Patient 5 0 0.0018 Patient 6 0 0.0028 Patient 7 0 0.0007
Patient 8 0 0.0057 Average 0.0000000 0.0025125
Expression Level of DNA of the Present Invention in Human
Peripheral Blood
[0185] The expression level of the G9 gene in peripheral blood of
normal individuals and patients with colorectal cancer was compared
using a quantitative real-time PCR method.
[0186] Blood was collected into a PAXgene blood RNA tube
(manufactured by PreAnalytix) from healthy volunteers and patients
with colorectal cancer. After the blood was mixed by inversion with
reagents in the tube and reacted at room temperature for 24 hours,
RNA was extracted with a PAXgene blood RNA kit (manufactured by
PreAnalytix). Using Super-Script First-Strand Synthesis System
(manufactured by Invitrogen), cDNA was synthesized with
accompanying random primers. This DNA was used as templates to
carry out quantitative real-time PCR with ABI PRISM 7700
(manufactured by Applied Biosystems Japan) using a 5' primer (SEQ
ID NO: 4), a 3' primer (SEQ ID NO: 5), and a TaqMan probe (SEQ ID
NO: 6). PCR conditions comprised reaction at 50.degree. C. for 2
minutes and 95.degree. C. for 10 minutes, followed by cycles
repeated 50 times, each cycle having 95.degree. C. for 15 seconds
and 60.degree. C. for 1 minute. The obtained measurement values
were divided by a value from .beta.-actin as an internal standard
gene quantified using a kit manufactured by Applied Biosystems
Japan Ltd., in order to correct variations among individuals.
Comparison was made between the normal individuals and the patients
with colorectal cancer.
[0187] The result has demonstrated that the transcription level of
the DNA of the present invention in the peripheral blood from the
patients with colorectal cancer is significantly greater than that
in the peripheral blood from the normal individuals. When patients
with colorectal cancer having the measurement value that exceeds
the average measurement value of normal individuals+(standard
deviation.times.2) were assessed to be positive, the positive rate
of the patients with colorectal cancer was 67% (see Table 4).
TABLE-US-00004 TABLE 4 Normal Patient with No. individual
colorectal cancer Assessment 1 90 121 Positive 2 107 68 Negative 3
82 199 Positive 4 81 418 Positive 5 87 123 Positive 6 92 Negative 7
196 Positive 8 86 Negative 9 473 Positive 10 267 Positive 11 110
Positive 12 46 Negative Average 89.4 183.3 Standard 9.4 132.1
deviation
Expression Level of DNA of the Present Invention in a Variety of
Human Normal Tissues
[0188] The expression level (at cDNA) of the DNA of the present
invention derived from human normal tissues was compared using a
quantitative real-time PCR method in the same way as above. RNA
from these tissues is commercially available from Clontech Inc.,
and so on. The synthesis of cDNA employed Super-Script First-Strand
Synthesis System (manufactured by Invitrogen). However, pCR2.1
(Invitrogen) DNA containing a GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) gene was used for creating the calibration curve of
an internal standard. The pBluescript(R) II SK(-) vector DNA
containing the ORF of the cloned G9 nucleic acid was used for
creating the calibration curve of the G9.
[0189] As a result, the expression of the DNA of the present
invention is observed in most of the human normal tissues. It has
revealed that the expression is relatively high in especially the
bone marrow, the spleen, and the small intestine and however, the
large intestine and the prostate have a considerably weak
expression or substantially no expression (see FIG. 1).
Example 3 Expression of Isolated Full-Length G9
[0190] The plasmid DNA where the G9 was incorporated into the
pBluescript(R) II SK(-) and pcDNA3.1 (manufactured by Invitrogen)
were individually treated with restriction enzymes HindIII and
EcoRI, with which a reaction solution was then mixed, followed by
ligation reaction to thereby introduce the ORF of the G9 into the
pcDNA3.1(+). The reaction solution was purified by an ethanol
precipitation method. Then, by investigating the sequence, the G9
was confirmed to be introduced into the pcDNA3.1(+) and this was
designated as pcDNA3.1(+)-G9. The pcDNA3.1(+)-G9 was mixed with a
competent cell (E. coli DH5.alpha.). The mixture was subjected to a
heat shock method (42.degree. C., 45 sec.) and seeded to a LB agar
medium containing ampicillin. The next day, a single colony was
further cultured to collect plasmid DNA. The collected plasmid DNA
was confirmed to contain the nucleic acid sequence of interest and
the nucleotide sequence was determined.
[0191] The pcDNA3.1(+/-) is an expression vector for a broad
variety of mammalian cells. It is a vector for forward
transcription, in which a sequence likely to form the secondary
structure of RNA is removed from a multi-cloning site (MCS)
sequence by improving conventional pcDNA3.1 for enhancing
expression level. The pcDNA3.1(+/-) has the enhancer/promoter of
CMV and allows a high level of expression. RNA is stabilized by a
polyadenylated signal and a transcription termination sequence.
Because there exists a SV40 origin, the pcDNA3.1(+/-) can be
replicated in a cell in which a SV40 Large T antigen is being
expressed. An ampicillin-resistant gene is introduced therein for
selection in E. coli. A neomycin-resistant gene is also introduced
therein for selection for producing a stable strain in a mammalian
cell.
[0192] HCT15 cells, human colorectal cancer-derived cell lines,
were used to conduct procedures below for creating G9-expressing
stable strains. The HCT15 cells were suspended at 2.times.10.sup.6
cells in 10 ml of a RPMI-1640 medium containing 10% fetal bovine
serum but no antibiotic, then seeded to a 10-cm dish, and cultured
in a CO.sub.2 incubator at 37.degree. C. for 16 hours. The plasmid
DNA (20 ng) of the pcDNA3.1(+)-G9 and 30 .mu.l of Lipofectamine
2000 (manufactured by Invitrogen) were mixed with 1.5 ml of
OPTI-MEM (manufactured by Invitrogen), respectively, and incubated
at room temperature for 5 minutes. Further, these two solutions
were gradually mixed and incubated at room temperature for 20
minutes. This mixture solution was added dropwise to the dish and
cultured in a CO.sub.2 incubator at 37.degree. C. for 48 hours. The
cells were subcultured by a standard method. On this occasion,
RPMI-1640 (manufactured by Invitrogen) was used as a medium, to
which fetal bovine serum, and penicillin (manufactured by
Invitrogen), streptomycin (manufactured by Invitrogen), and
Geneticin (neomycin; manufactured by Invitrogen) as antibiotics
were added. Because cells in which the pcDNA3.1(+)-G9 has not been
introduced are allowed to die out by the addition of Geneticin,
continued culture results in the survival of only cells in which
the pcDNA3.1(+)-G9 has been introduced. These cells were used as
G9-expressing stable strains.
Expression of G9 Recombinant Protein in Mammalian Cell Line
[0193] For obtaining a G9 recombinant protein, the G9 was expressed
in a human kidney-derived cell line 293T. Only the expression of an
active region from the 105th amino acid to the C terminus in SEQ ID
NO: 2 which relatively retains homology to at least .beta.1,3GlcNAc
transferase and .beta.1,3Gal transferase may be adequate for
investigating a function. However, here, it has been decided to
express two predicted active regions from the 24th amino acid and
the 33rd amino acid to the C terminus in the G9.
[0194] Using, as a template, the plasmid DNA where the G9 was
incorporated into the pBluescript(R) II SK(-), PCR reaction was
carried out with each of 5' primers (SEQ ID NOs: 8 and 9) and a 3'
primer (SEQ ID NO: 10) to obtain a DNA fragment of interest. The
PCR method comprised 25 cycles, each cycle having 94.degree. C. for
30 seconds, 65.degree. C. for 1 minute, and 72.degree. C. for 1
minute. Then, a PCR product was subjected to agarose gel
electrophoresis. The gel was cut out by a gel excision method to
isolate the PCR product by a standard method. This PCR product had
HindIII on the 5' side and EcoRI on the 3' side as restriction
sites. This DNA fragment and pFLAG-CMV3 were individually treated
with restriction enzymes HindIII and EcoRI, with which a reaction
solution was then mixed, followed by ligation reaction to thereby
introduce the DNA fragment into the pFLAG-CMV3. The reaction
solution was purified by an ethanol precipitation method and then
mixed with a competent cell (E. coli DH5.alpha.). The mixture was
subjected to a heat shock method (42.degree. C., 45 sec.) and
seeded to a LB agar medium containing ampicillin.
[0195] The next day, the DNA of interest in the resulting colony
was directly confirmed by PCR. After the DNA sequence was confirmed
by sequencing for additional confirmation, the vector (pFLAG-CMV3)
was extracted and purified.
[0196] Human kidney cell-derived cell line 293T cells were
suspended at 2.times.10.sup.6 cells in 10 ml of a DMEM medium
containing 10% fetal bovine serum but no antibiotic, then seeded to
a 10-cm dish, and cultured in a CO.sub.2 incubator at 37.degree. C.
for 16 hours. The pFLAG-CMV3-G9 (20 ng) and 30 .mu.l of
Lipofectamine 2000 (manufactured by Invitrogen) were mixed with 1.5
ml of OPTI-MEM (manufactured by Invitrogen), respectively, and
incubated at room temperature for 5 minutes. Further, these two
solutions were gradually mixed and incubated at room temperature
for 20 minutes. This mixture solution was added dropwise to the
dish and cultured in a CO.sub.2 incubator at 37.degree. C. for 48
hours.
[0197] With 10 ml of the resulting culture supernatant, NaN.sub.3
(0.05%), NaCl (150 ml), CaCl.sub.2 (2 ml), anti-FLAG M1 affinity
gel (manufactured by Sigma) (100 .mu.l) were mixed and stirred
overnight at 4.degree. C. The next day, the mixture was centrifuged
(3000 rpm, 5 min, 4.degree. C.) to collect a pellet to which 900
.mu.l of 2 ml CaCl.sub.2/TBS was in turn added. The mixture was
centrifuged again (2000 rpm, 5 min, 4.degree. C.), and the
resulting pellet was floated in 200 .mu.l of 1 ml CaCl.sub.2/TBS
and used as a sample (G9 enzyme solution) for activity measurement.
A portion of this was subjected to electrophoresis by SDS-PAGE and
subsequently to western blotting with anti FLAG-M2 peroxidase
(manufactured by Sigma), to confirm the expression of the G9
protein of interest. As a result, a band was detected at the
position of approximately 45 kDa, and the expression was therefore
confirmed.
Expression of G9 Recombinant Protein in an Insect Cell Line
[0198] For obtaining a G9 recombinant protein, the G9 was expressed
in an insect cell. Only the expression of an active region from the
105th amino acid to the C terminus in SEQ ID NO: 2 which relatively
retains homology to at least .beta.1,3GlcNAc transferase and
.beta.1,3Gal transferase may be adequate for investigating a
function. However, here, it has been decided to express a predicted
active region from the 36th amino acid to the C terminus in the
G9.
[0199] Using, as a template, the plasmid DNA where the G9 was
incorporated into the pBluescript(R) II SK(-), PCR reaction was
carried out with a 5' primer (SEQ ID NO: 11) and a 3' primer (SEQ
ID NO: 12) to obtain a DNA fragment of interest. The PCR method
comprised 25 cycles, each cycle having 94.degree. C. for 30
seconds, 65.degree. C. for 1 minute, and 72.degree. C. for 1
minute. Then, a PCR product was subjected to agarose gel
electrophoresis. The gel was cut out by a gel excision method to
isolate the PCR product by a standard method. The PCR product thus
isolated was incorporated into pDONR.TM. 201 (manufactured by
Invitrogen) by BP clonase reaction to create an "entry clone".
[0200] Reaction was carried out by incubating 2 .mu.l of the PCR
product, 1 .mu.l (150 ng) of the pDONR 201, 2 .mu.l of a BP
reaction buffer, 3 .mu.l of a Tris-EDTA buffer (pH 8.0;
hereinafter, also abbreviated to "TE"), and 2 .mu.l of BP clonase
mix at 25.degree. C. for 1 hour. The mixture was then supplemented
with 1 .mu.l of a proteinase K (manufactured by Kaken
Pharmaceutical) and incubated at 37.degree. C. for 10 minutes to
terminate the reaction. The reaction mixture solution (11 .mu.l)
was mixed with 100 .mu.l of a competent cell (E. coli DH5.alpha.),
then transformed by a heat shock method, and seeded to a LB plate
containing kanamycin. The next day, a colony was collected to
confirm the introduction of the DNA of interest and its nucleotide
sequence by PCR. The vector in which the DNA was inserted
(pDONR-G9) was extracted and purified according to a standard
method. The nucleotide sequence of the DNA inserted in this vector
was confirmed to contain the nucleotide sequence described in SEQ
ID NO: 1.
Preparation of Expression Clone
[0201] The above-described entry clone has attL sites at both ends
of the inserted site that are recombination sites when a .lamda.
phage is excised from E. coli. By mixing the entry clone with a LR
clonase (mixture of .lamda. phage recombinases Int, IHF, and Xis)
and a destination vector (which has attR), the inserted site was
transferred to the destination vector to generate an expression
clone.
[0202] With 1 .mu.l of the entry clone (pDONR-G9), 0.5 .mu.l (75
ng) of the destination vector (pFBIF), 2 .mu.l of a LR reaction
buffer, 4.5 .mu.l of TE, and 2 .mu.l of LR clonase mix (mixture
solution of .lamda. phage recombinases Int, IHF, and Xis) were
incubated at 25.degree. C. for 1 hour. The mixture was then
supplemented with 1 .mu.l of a proteinase K (manufactured by Kaken
Pharmaceutical) and incubated at 37.degree. C. for 10 minutes to
terminate the reaction (this recombination reaction yields
pFBIF-G9). The pFBIF was obtained by inserting an Ig.kappa. signal
sequence and a FLAG peptide for purification into pFastBac1
(manufactured by Invitrogen) according to a standard method. For
further inserting the Gateway sequence (attR) into the pFBIF,
Gateway Vector Conversion System (manufactured by Invitrogen) was
used to insert a conversion cassette. This conversion cassette is a
cassette for altering an expression vector to a destination vector
and has an attR recombination site, a chloramphenicol-resistant
gene, and a ccdB gene encoding a protein that inhibits an E. coli
DNA gyrase. The Ig.kappa. signal sequence was inserted for
rendering an expressed protein secretory, while the FLAG tag was
inserted for facilitating purification.
[0203] The reaction mixture solution (11 .mu.l) containing the
pFBIF-G9 and 100 .mu.l of a competent cell E. coli DH5.alpha. were
mixed and transformed by a heat shock method, and the resulting
recombinant DH5.alpha. was seeded to a LB medium containing
ampicillin and then cultured. After 24-hour culture, a colony was
collected, and the plasmid (pFBIF-G9) was extracted and purified by
QIAprep Spin Miniprep Kit (manufactured by Qiagen). A PCR method
was used to confirm the insertion of the DNA of interest.
Preparation of Bacmid by Bac-to-Bac System (Manufactured by
Invitrogen)
[0204] Subsequently, using a Bac-to-Bac system (manufactured by
Invitrogen), recombination was conducted between the pFBIF-G9 and a
bacmid to insert the G9 sequence into the bacmid capable of
proliferation in an insect cell. This system is a system that
utilizes the recombination site of Tn7 and allows the incorporation
of the gene of interest (G9) into the bacmid through a
recombination protein produced by a helper plasmid only by
introducing, into bacmid-containing E. coli (E. coli DH10Bac.TM.),
pFastBac in which the gene of interest is inserted (i.e.,
pFBIF-G9). Moreover, the bacmid contains a LacZ gene and is
selectable by classical colony colors (blue (without insertion) to
white (with insertion)).
[0205] That is, 50 .mu.l of the above-described purified vector
(pFBIF-G9) and 50 .mu.l of a competent cell (E. coli DH10Bac) were
mixed, then transformed by a heat shock method, and seeded to a LB
medium containing kanamycin, gentamicin, tetracycline,
5-bromoindolyl-.beta.-D-galactopyranoside (Bluo-gal), and
isopropyl-.beta.-D-thiogalactopyranoside (IPTG). After 24 hours, an
independent white colony where the DNA of interest is inserted into
the bacmid was collected and further cultured to collect the bacmid
according to a standard method.
Introduction of Bacmid into Insect Cell
[0206] The insertion of the DNA of interest into the collected
bacmid was confirmed according to a standard method, and the bacmid
was transfected into an insect cell (Sf21; manufactured by
Invitrogen). That is, a Sf900SFM medium (manufactured by
Invitrogen) containing an antibiotic was added to the Sf21 cells at
9.times.10.sup.5 cells/2 ml which was then allowed to adhere to a
35-mm Petri dish at 27.degree. C. for 1 hour. After the cells were
confirmed to adhere to the Petri dish, the culture solution was
aspirated. The cells were supplemented and incubated at 27.degree.
for 5 hours with a culture solution where 800 .mu.l of Sf900II
(manufactured by Invitrogen) was added to a solution of lipid-DNA
complexes (solution obtained by gently mixing and incubating, at
room temperature for 30 minutes, A Solution (mixture of 5 .mu.l of
the above-described bacmid added to 100 .mu.l of Sf-900SFM) and B
Solution (mixture of 6 .mu.l of Cellfectin Reagent (manufactured by
Invitrogen) added to 100 .mu.l of Sf-900SFM)). Then, the medium was
removed, and the cells were supplemented with 2 ml of a SF900SFM
medium containing an antibiotic and incubated 27.degree. C. for 72
hours. After culture, the cells were liberated by pipetting. The
cells and the culture solution were collected and centrifuged at
1000.times.g for 10 minutes to collect a supernatant (this
supernatant was used as a "primary virus solution").
[0207] Further, 1.times.10.sup.7 Sf21 cells/20 ml of Sf-900SFM
(containing an antibiotic) were added to a T75 culture flask and
incubated at 27.degree. C. for 1 hour. After the adhesion of the
cells to the flask, 800 .mu.l of the primary virus solution was
added and cultured at 27.degree. C. for 48 hours. After culture,
the cells were liberated by pipetting. The cells and the culture
solution were collected and centrifuged at 1000.times.g for 10
minutes to collect a supernatant (this supernatant was used as a
"secondary virus solution").
[0208] Further, 1.times.10.sup.7 Sf21 cells/20 ml of Sf-900SFM
(containing an antibiotic) were added to a T75 culture flask and
incubated at 27.degree. C. for 1 hour. After the adhesion of the
cells to the flask, 1000 .mu.l of the secondary virus solution was
added and cultured at 27.degree. C. for 84 hours. After culture,
the cells were liberated by pipetting. The cells and the culture
solution were collected and centrifuged at 1000.times.g for 10
minutes to collect a supernatant (this supernatant was used as a
"tertiary virus solution").
[0209] In addition, 100 .mu.l of Sf-900SFM (containing an
antibiotic) containing Sf21 cells at a concentration of
6.times.10.sup.5 cells/ml and subsequently 1 ml of the tertiary
virus solution were added to a 100-ml spinner flask and cultured at
27.degree. C. for 96 hours. After culture, the cells and the
culture solution were collected and centrifuged at 1000.times.g for
10 minutes to collect a supernatant (this supernatant was used as a
"quaternary virus solution").
[0210] NaN.sub.3, NaCl, and CaCl.sub.2 were added to 10 ml of the
quaternary virus solution. The final concentration is set to 0.05%
for NaN.sub.3, to 150 mM for NaCl, and to 2 mM for CaCl.sub.2. To
the mixture solution, 50 .mu.l of anti-FLAG M1 antibody affinity
gel (manufactured by Sigma) was added and gently mixed by inversion
at 4.degree. C. for 16 hours. Following centrifugation
(1000.times.g, 3 min. 4.degree. C.) to remove the resulting
supernatant, the affinity gel was washed twice with TBS
(Tris-buffered saline, pH 7.4) containing 1 mM CaCl.sub.2. After
washing, the affinity gel was suspended in 200 .mu.l of TBS (pH
7.4) containing 1 mM CaCl.sub.2, and this suspension was used as a
G9 enzyme solution for activity measurement.
Example 4 Construction of G9 Enzyme Protein with Mammal Cell
Expression System
(1) Construction of Secretory G9 Polypeptide Recombinant
[0211] As shown in the examples above, the G9 polypeptide
constructed by deleting a region on the N-terminal side of the
polypeptide was confirmed to be capable of being expressed as a
protein in an insect cell and so on. A protein constructed as a
FLAG peptide-fused G9 polypeptide by deleting a region on the
N-terminal side in a similar way can also be isolated and purified
as an enzyme protein having an activity by a mammalian cell
expression system.
[0212] The part likely to be the catalytic region of an enzyme
protein in the ORF of the G9 nucleic acid could be obtained by
using, as a template, the plasmid DNA where the G9 is incorporated
into the pBluescript(R) II SK(-) to carry out PCR reaction with a
5' primer having a nucleic acid sequence of either SEQ ID NO: 13 or
14 and a 3' primer having a nucleic acid sequence of SEQ ID NO: 15.
The PCR method employed a Pfx Taq DNA polymerase (manufactured by
Invitrogen) and comprised 25 cycles, each cycle having 94.degree.
C. for 15 seconds, 60.degree. C. for 30 seconds, and 68.degree. C.
for 1 minute in the presence of 5 ng of the template in a total of
50 .mu.l of a reaction solution. Then, a PCR product was subjected
to agarose gel electrophoresis. The gel was cut out by a gel
excision method to isolate the PCR product by a standard
method.
[0213] A recombinant DNA fragment having a nucleic acid sequence
from nucleotide Nos. 76 to 1194 in SEQ ID NO: 1 was obtained by PCR
reaction using the 5' primer of SEQ ID NO: 13 and the 3' primer of
SEQ ID NO: 15. This fragment corresponds to that encoding an amino
acid sequence from amino acid Nos. 26 to 397 in SEQ ID NO: 2, that
is, an amino acid sequence of SEQ ID NO: 16.
[0214] Alternatively, a recombinant DNA fragment having a sequence
shorter than that of the above-described DNA fragment was obtained
by PCR reaction using the 5' primer of SEQ ID NO: 14 and the 3'
primer of SEQ ID NO: 15. This fragment is composed of a nucleotide
sequence from nucleotide Nos. 97 to 1194 in SEQ ID NO: 1 and
corresponds to that encoding an amino acid sequence from amino acid
No. 33 to 397 in SEQ ID NO: 2, that is, an amino acid sequence of
SEQ ID NO: 17. Although all of the polypeptides expressed from
these recombinant DNA fragments were confirmed to have an enzyme
activity, the longer DNA fragment obtained by the combination of
the primers of SEQ ID NO: 13 and SEQ ID NO: 15 was used in the
experiments below.
[0215] By adding the signal sequence of preprotrypsin and a FLAG
peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) to the region of the G9
polypeptide as described above in which initiation methionine has
been removed, the secretion and expression of a FLAG peptide-fused
G9 polypeptide can be undertaken. That is, after the DNA fragment
amplified with the primers was treated with restriction enzymes
HindIII and EcoRI, the DNA was purified and inserted between
HindIII and EcoRI in the cloning site of a pFLAG-CMV3 (manufactured
by Invitrogen, Carlsbad, Calif.) vector, followed by ligation
reaction to thereby create pFLAG-CMV3-G9. The reaction solution was
purified by an ethanol precipitation method and then mixed with a
competent cell (E. coli DH5.alpha.). The mixture was subjected to a
heat shock method (42.degree. C., 45 sec.) and seeded to a LB agar
medium containing ampicillin. The next day, the insertion of the
DNA of interest in the resulting colony was directly confirmed by
PCR. After the plasmid DNA sequence was confirmed by sequencing for
additional confirmation, the vector (pFLAG-CMV3-G9) was extracted
and purified. As described below, the obtained recombinant DNA can
be expressed as an enzyme protein and purified, and its activity
can be confirmed. Here, computer prediction and preliminary
experiments, and so on indicated that catalytic regions in both
transmembrane and secretory forms probably had the same exon
(sequence). In addition, the N-terminal side is composed of a
region not required for the temporary confirmation of an activity
such as a transmembrane region, so that the sequence portion as
described above was used.
[0216] The pFLAG-CMV3-G9 plasmid created as above was transfected
into a HEK293T cell, and the FLAG peptide-fused G9 polypeptide was
expressed and secreted into a culture supernatant. The transfection
method was carried out using Lipofectamine 2000 (manufactured by
Invitrogen) according to the attached instruction. Specifically,
human kidney cell-derived cell line 293T cells were suspended at
2.times.10.sup.6 cells in 10 ml of a DMEM medium containing 10%
fetal bovine serum but no antibiotic, then seeded to a 10-cm dish,
and cultured in a CO.sub.2 incubator at 37.degree. C. for 16 hours.
The pFLAG-CMV3-G9 (20 ng) and 30 .mu.l of Lipofectamine
2000-(manufactured by Invitrogen) were mixed with 1.5 ml of
OPTI-MEM (manufactured by Invitrogen), respectively, and incubated
at room temperature for 5 minutes. Further, these two solutions
were gradually mixed and incubated at room temperature for 20
minutes. This mixture solution was added dropwise to the dish and
cultured in a CO.sub.2 incubator at 37.degree. C. for 48 hours.
[0217] It is noted that the portion of 372 amino acid residues
described in SEQ ID NO: 16 is the portion free from the FLAG and so
on in the amino acid sequence of the G9 polypeptide portion
obtained as described above. On the other hand, a polypeptide
portion of SEQ ID NO: 17 is obtained in a similar way, but having a
slightly shorter length is contained in the sequence of SEQ ID NO:
16 and is a region consisting of 282 amino acid residues containing
a region that is considered as an active domain from the viewpoint
of homology, to a .beta.1,3-N-acetyl-D-glucosaminyltransferase
protein known in the art. Those containing at least this region
probably show the enzyme activity of interest.
(2) Purification of G9 Polypeptide Secreted into Culture
Solution
[0218] The G9 polypeptide recombinant described above is secreted
and expressed as a fusion protein with the FLAG peptide and as
such, can easily be purified using anti-FLAG M1 antibody affinity
gel (manufactured by Sigma). The purified enzyme protein may be
liberated from the anti-FLAG M1 affinity gel or otherwise may be
used by being adsorbed in the gel. In the present example, the
enzyme was used by being absorbed in the gel, to carry out
experiments.
[0219] NaN.sub.3, NaCl, and CaCl.sub.2 were added to 15 ml of the
culture supernatant acquired as above to be brought to 0.1%, 150
mmol/l, and 2 mmol/l, respectively, at the final concentration. The
mixture was then supplemented with 100 .mu.l of Anti-FLAG M1
Affinity Gel (manufactured by COSMO Bio) and gradually stirred
overnight at 4.degree. C.
[0220] The next day, the mixture was centrifuged (3000 rpm, 5 min,
4.degree. C.) to collect a pellet to which 900 .mu.l of 2 mM
CaCl.sub.2/TBS was in turn added. The mixture was centrifuged again
(2000 rpm, 5 min, 4.degree. C.), and the resulting pellet was
floated in 200 .mu.l of 1 ml CaCl.sub.2/TBS and used as a sample
(G9 enzyme solution) for activity measurement. A portion of this
was subjected to electrophoresis by SDS-PAGE and subsequently to
western blotting with anti-FLAG M2 peroxidase (manufactured by
Sigma) to confirm the expression of the G9 protein of interest. As
a result, a band was detected at the position of approximately 45
kDa, and the expression was therefore confirmed.
[0221] After washing, the gel was supplemented with 30 .mu.l of a
buffer containing 50 mmol/l Tris-HCl (pH 7.4), 150 mmol/l NaCl, and
2 mmol/l EDTA and treated at 4.degree. C. for 30 minutes to thereby
elute the protein adsorbed in the gel. Then, the 10-minute
centrifugation of the gel at 160.times.g yielded a supernatant.
After the gel was supplemented again with 30 .mu.l of a buffer
containing 50 mmol/l Tris-HCl (pH 7.4), 150 mmol/l NaCl, and 2
mmol/l EDTA and treated at 4.degree. C. for 10 minutes, the gel was
centrifuged at 160.times.g for 10 minutes to thereby acquire a
supernatant. Then, the above procedures were carried out again, and
a total of 3 elution procedures were performed. The obtained eluate
was supplemented with 1 mol/l CaCl.sub.2 to be brought to 4 mmol/l
at the final concentration. This eluate was used as an enzyme
source.
Example 5 Analysis of G9 Enzyme Protein for Enzyme Activity
(Activity Measurement Using Glycolipid and Synthetic
Monosaccharides, and so on, as Substrates
[0222] The result of comparison with other glycosyltransferases
known in the art on the basis of the amino acid sequence described
in SEQ ID NO: 2 has suggested that the G9 is classified into
transferases in light of, for example, the conservation of a
sequence in the C-terminal region considered as an active site.
Therefore, for example, UDP-GlcNAc can be used as a GlcNAc donor
substrate to confirm the enzyme activity of the enzyme solution
containing the G9 polypeptide, which has been obtained in Example 4
above.
[0223] In the present example, the activity of the G9 enzyme was
measured according to almost the same method as methods described
in the following references [1] to [3]: [0224] [1] Shiraishi N,
Natsume A, Togayachi A, Endo T, Akashima T, Yamada Y, Imai N,
Nakagawa S, Koizumi S, Sekine S, Narimatsu H, Sasaki K.,
Identification and characterization of three novel beta
1,3-N-acetylglucosaminyltransferases structurally related to the
beta 1,3-galactosyltransferase family., J Biol. Chem., 2001 Feb. 2;
276(5): 3498-507; [0225] [2] Togayachi A, Akashima T, Ookubo R,
Kudo T, Nishihara S, Iwasaki H, Natsume A, Mio H, Inokuchi J,
Irimura T, Sasaki K, Narimatsu H., Molecular cloning and
characterization of UDP-GlcNAc:lactosylceramide beta
1,3-N-acetylglucosaminyltransferase (beta 3Gn-T5), an essential
enzyme for the expression of HNK-1 and Lewis X epitopes on
glycolipids., J Biol chem., 2001 Jun. 22; 276(25): 22032-40; and
[0226] [3] Iwai T, Inaba N, Naundorf A, Zhang Y, Gotoh M, Iwasaki
H, Kudo T, Togayachi A, Ishizuka Y, Nakanishi H, Narimatsu H.,
Molecular cloning and characterization of a novel
UDP-GlcNAc:GalNAc-peptide .beta. 1,3-N
acetylglucosaminyltransferase (.beta.3Gn-T6), an enzyme
synthesizing the core 3 structure of O-glycans., J Biol. Chem.,
2001 Jun. 22; 276(25): 22032-40.
(1) Investigation of Substrate Specificity
[0227] The FLAG peptide-fused G9 polypeptide obtained in the
example above was measured for a
.beta.1,3-N-acetylglucosaminyltransferase activity with
monosaccharide/oligosaccharide/glycolipid/glycoprotein as
substrates according to known methods (e.g., the above-described
references and FEBS, 462, 289 (1999), J. Biol. Chem. 269,
14730-14737 (1994), J. Biol. Chem., 267, 23507 (1992), and J. Biol.
Chem., 267, 2994 (1992)).
[0228] pNp-.alpha.-Glc, pNp-.beta.-Glc, pNp-.alpha.-GlcNAc,
pNp-.beta.-GlcNAc, pNp-.alpha.-Gal, oNp-.beta.-Gal,
pNp-.alpha.-GalNAc, pNp-.alpha.-Xyl, oNp-.beta.-Xyl,
pNp-.alpha.-Fuc, Bz-.alpha.-Man, Bz-.alpha.-ManNAc,
core1-.alpha.-pNp, core3-.alpha.-pNp, Bz-.beta.-lactoside,
GlcNAc.beta.1-4GlcNAc.beta.-Bz, and Gal.beta.1-4GlcNAc.alpha.-pNp
were used as acceptor substrates (see Table 5). These substrates
can be purchased from, for example, Sigma Corp. or Toronto Research
Chemicals Inc.
TABLE-US-00005 TABLE 5 Substrate specificity of G9 Acceptor
substrate Relative activity % 1 Gal.alpha.-pNP.sup.a ND 2
Gal.beta.-oNP.sup.a ND 3 GlcNAc.alpha.-Bz.sup.b ND 4
GlcNAc.beta.-Bz.sup.b ND 5 GalNAc.alpha.-pNP.sup.a ND 6
GalNAc.beta.-pNP.sup.b ND 7 Glc.alpha.-pNP.sup.a ND 8
Glc.beta.-pNP.sup.a ND 9 Fuc.alpha.-pNP.sup.a ND 10
Xyl.alpha.-pNP.sup.c ND 11 Xyl.beta.-pNP.sup.b ND 12
Man.alpha.-Bz.sup.c ND 13 Lactoside .beta.-Bz.sup.b 27 14
Gal.beta.1-3GalNAc.alpha.-pNP (core 1).sup.c ND 15
GlcNAc.beta.1-3GalNAc.alpha.-pNP (core 2).sup.c ND 16
Gal.beta.1-3GlcNAc.beta.-pNp.sup.a ND 17
GlcNAc.beta.1-4GlcNAc.beta.-Bz.sup.c ND 18
Gal.beta.1-4GlcNAc.alpha.-pNP.sup.c 100
(In Table 5, ND represents "not detected"; and the acceptor
substrates marked by the superscript "a" were purchased from
Calbiochem, the acceptor substrates marked by "b" were purchased
from Sigma-Aldrich Corp, and the acceptor substrates marked by "c"
were purchased from Toronto Research Chemicals Inc.)
[0229] A basic reaction solution in the case of using a
radiolabeled substrate as the donor substrate comprises 20 .mu.l in
total of 14 mM HEPES buffer (pH 7.4), 10 mM MnCl.sub.2, 0.15%
Triton CF-54, 0.75 mM ATP, 50 .mu.M UDP-GlcNAc (manufactured by
Sigma), 4.5 .mu.M (50 nCi) [.sup.14C]UDP-GlcNAc (manufactured by
Amersham Biosciences), 10 .mu.M substrate (each of the
above-described acceptor substrates), and an appropriate amount (5
to 10 .mu.l) of the purified enzyme protein (enzyme source obtained
in Example 4). Enzyme reaction was carried out at 37.degree. C. for
several hours up to 16 hours (usually for 16 hours).
[0230] After the completion of reaction, the resulting solution was
supplemented with 200 .mu.l of 0.1M KCl and lightly centrifuged,
and then a supernatant was obtained. After being washed once with
10 ml of methanol, the supernatant was loaded onto Sep-Pak C18
Cartridge (Waters) equilibrated by two-time washing with 10 ml of
0.1 M KCl, and the substrate and a product in the supernatant were
adsorbed in the cartridge. After the cartridge was washed twice
with 1 ml of pure water for HPLC, the adsorbed substrate and
product were eluted with 1 ml of methanol. Following the mixing
with a liquid scintillator (ACSII; manufactured by Amersham
Biosciences), the amount of radiation from the product was measured
with a scintillation counter.
[0231] As a result, the G9 polypeptide exhibited the highest
transferring activity for Gal.beta.1-4GlcNAc.alpha.-pNp of the
acceptor substrates tested and also exhibited a certain amount of
transferring activity for Bz-.beta.-lactoside (see Table 5).
(2) Investigation of Enzyme Reaction Conditions
[0232] Buffer conditions were investigated using the same reaction
solution and reaction time as above but using HEPES (pH 6.75 to
7.4) and sodium cacodylate (pH 6.4 to 7.2) instead of 14 mM HEPES
buffer (pH7.4) (see FIG. 2A).
[0233] As shown in FIG. 2A, the G9 polypeptide exhibited an
activity in the case of using any of sodium cacodylate and HEPES.
Generally, its activity was high at or around neutral. The G9
polypeptide had an activity that rises with an increase in pH
within the neutral region from pH 6.4 to 7.2 in the sodium
cacodylate and showed the maximum activity around pH 7.0 in the
HEPES.
[0234] Since some glycosyltransferases require a metal ion, the
metal ion requirement of the G9 enzyme was investigated. The same
reaction solution and reaction time as above were used, and
MnCl.sub.2, CoCl.sub.2, MgCl.sub.2, ZnCl.sub.2, NiCl.sub.2, and
CdCl.sub.2 were used instead of 10 mM MnCl.sub.2. The
concentrations of each of the metal ions were set to 2.5, 10, and
40 mM, and the respective enzyme reactions were conducted (see FIG.
2B).
[0235] As shown in FIG. 2B, the activity of the G9 polypeptide is
significantly enhanced in the presence of at least the Mn ion or Co
ion of the divalent metal ions and particularly remarkably enhanced
in the presence of the Mn ion. These enhanced activities rapidly
rise in the low concentration region of the ion and subsequently
gradually decrease. Although the Cd and Ni ions show slight
enhancing effect in the low concentration region, no enhancing
effect is substantially observed in the Mg and Zn ions.
[0236] The above results have demonstrated that the activity is
shown most highly under the basic reaction conditions used in (1)
above. The same conditions were used to carry out reaction in
experiments below.
(3) Activity Measurement in the Case of Using Oligosaccharides
Pyridylaminated with 2-aminopyridine (N-glycans) as Acceptor
Substrates
[0237] Commercially-available PA-oligosaccharides were used as
substrates. An oligosaccharide can be pyridylaminated (PA) with
2-aminopyridine according to a standard method (Hase, S., Ibuki,
T., and Ikenaka, T., J. Biochem. 95, 197-203 (1984)), and the
PA-oligosaccharide was purchased from TAKARA SHUZO Co., Ltd. or
SEIKAGAKU Corp. The specific test method is performed as
follows:
[0238] After 16-hour reaction at 37.degree. C. in a total of 20
.mu.L of reaction solution containing 14 mM HEPES buffer (pH 7.4),
50 mM UDP-GlcNAc, 10 mM MnCl.sub.2, 0.15% Triton CF-54, 40 pmol
acceptor substrate (PA-oligosaccharide), and an appropriate amount
(200 ng) of a purified enzyme protein, a product was detected by
high performance liquid chromatography (HPLC; described below in
detail). The enzyme purified with the anti-FLAG M1 antibody
affinity gel in Example 4 was used as the above-described purified
enzyme protein.
[0239] After the assay solution where the reaction was completed
was treated at 100.degree. C. for 5 minutes, the solution was
supplemented with 80 .mu.l of pure water for HPLC and centrifuged
at 10,000.times.g for 5 minutes to obtain a supernatant.
Subsequently, the supernatant was loaded onto an Ultrafree-MC
column (manufactured by Millipore), and a portion (40 .mu.l)
thereof was subjected to HPLC. The Ultrafree-MC column was used
according to the attached instruction.
[0240] HPLC was carried out using a PALPAK Type R column (TAKARA)
as a column and Eluant A: 100 mM acetic acid/triethylamine (pH 4.0)
and Eluant B: 100 mM acetic acid/triethylamine (pH 4.0)/0.5%
1-butanol as eluants under such conditions that: Eluant B gradient
was 5 to 55% (0 to 60 minutes); a column temperature was 40.degree.
C.; and a flow rate was 1 ml/min. A product was detected using a
fluorescence spectrum photometer RF-10AXL (manufactured by
Shimadzu) (excitation wavelength at 320 nm and radiation wavelength
at 400 nm).
[0241] As a result, the G9 polypeptide exhibited a significant
activity for tetraantennary N-glycans (PA-004 and PA-011: Takara
PA-substrate number) (see FIG. 3). By contrast, a
.beta.1,3-N-acetylglucosaminyltransferase 2 (.beta.3GnT2) known in
the art exhibits an activity for all types (PA-001 to -0011) of
N-glycans, regardless of the number of an oligosaccharide. This
also suggests that the G9 polypeptide in the present example has a
selective activity, that is, it exhibits a significant activity for
a tetraantennary N-glycan but exhibits no significant activity for
monoantennary to triantennary N-glycans. In the result shown in
FIG. 3, the activity of the G9 polypeptide appears relatively weak
as compared to that of the
.beta.1,3-N-acetylglucosaminyltransferase 2. However, this is
because the .beta.1,3-N-acetylglucosaminyltransferase 2 is a
homolog enzyme having a greatly strong enzyme activity.
(4) Activity Measurement in the Case of Using Glycoproteins as
Acceptor Substrates
[0242] Enzyme reaction was carried out with glycoproteins as
substrates. A reaction solution comprised 20 .mu.l in total of 14
mM HEPES buffer (pH 7.4), 10 mM MnCl.sub.2, 0.15% Triton CF-54,
0.75 mM ATP, 50 .mu.M UDP-GlcNAc (manufactured by Sigma), 4.5 .mu.M
(50 nCi) [.sup.14C]UDP-GlcNAc (manufactured by Amersham
Biosciences), 40 .mu.l of an acceptor substrate, and an appropriate
amount (5 to 10 .mu.l) of a purified enzyme protein. A
.alpha.1-acid glycoprotein (orosomucoid; manufactured by Sigma),
ovalbumin (manufactured by Sigma), or ovomucoid (manufactured by
Sigma) was used as the above described acceptor substrate. The
enzyme purified with the anti-FLAG M1 antibody affinity gel in
Example 4 was used as the above-described purified enzyme
protein.
[0243] Reaction was carried out at 37.degree. C. for several hours
up to 16 hours. A portion of a reaction product was subjected to
enzyme digestion with glycopeptidase F (GPF; manufactured by
TAKARA) according to the instruction. The samples both before and
after enzyme digestion were analyzed by 10% SDS-PAGE (see FIG.
4).
[0244] As a result, the G9 polypeptide showed a GlcNAc-transferring
activity for all of the glycoproteins (relatively weak activity for
the ovalbumin) and the band disappeared by GPF digestion. This has
demonstrated that such transferring reaction takes place against
the N-glycan oligosaccharide of each glycoprotein.
Example 6 Construction of G9 Knockout Mouse
[0245] At least the ORF of an mG9 gene is likely to have a single
exon. Created is a targeting vector (pBSK-mG9-KOneo) in which a
chromosome fragment (approximately 10 kb) mainly having an
approximately 10-kb fragment containing a region considered as the
active domain of the mG9 desired to be knocked out (e.g., a
nucleotide sequence from nucleotide No. 97 to 1194 in SEQ ID NO: 1)
was inserted into pBluescript(R) II SK (-) (manufactured by
TOYOBO). The pBSK-mG9-KOneo employs neo (neomycin-resistant gene)
as a drug-resistant gene and lacks the predicted
GlcNAc-transferring activity region of the mG9, and this lacking
portion is replaced with the neo. After the pBSK-mG9-KOneo thus
obtained is rendered linear with a restriction enzyme NotI, its
80-.mu.g aliquot is transfected (e.g. electroporated) into ES cells
(derived from an E14/129Sv mouse) to pick G418-resistant colonies.
The G418-resistant colonies are transferred to a 24-well plate and
cultured. After some of the cells are cryopreserved, DNA is
extracted from the remaining ES cells, and approximately 120
colonies of clones that have undergone recombination are selected
by PCR. In addition, PCR and southern blotting, and so on, are used
to ascertain that recombination occurs as planned. Ultimately, 10
clones of recombinants are selected. The ES cells from two of the
selected clones are injected into the blastocyst of a C57BL/6
mouse. The murine embryo in which the ES cell has been injected is
transplanted into the uterus of a foster mother mouse to generate a
chimeric mouse. Then, a hetero-knockout mouse can be obtained by
germ transmission.
Sequence CWU 1
1
2011194DNAHomo sapiens 1atgcgctgcc ccaagtgcct tctctgcctg tcagcactgc
tcacactcct gggcctcaaa 60gtgtacatcg agtggacatc cgagtcccgg ctcagcaagg
cctaccccag ccctcggggc 120accccgccaa gccccacgcc agccaaccct
gagcccaccc tacctgccaa cctctccacc 180cgcctgggcc agactatccc
gctgcccttt gcttactgga accagcagca gtggcggctg 240gggtccctgc
ccagtgggga cagcactgaa acggggggct gccaggcttg gggggccgcc
300gccgccaccg agatccctga cttcgcctcc taccccaagg acctccgccg
cttcttgctg 360tcagcagcct gccggagctt cccacagtgg ctgcctggag
gtggtggcag ccaagtctcc 420agctgctcag atactgatgt cccctacctg
ctgttggccg tcaagtcaga accagggcgc 480tttgcagaac gacaggccgt
gagagagacg tggggcagtc cagctccagg gatccggctg 540ctcttcctgc
tagggtctcc ggtgggtgag gcggggcctg acctagactc actagtggcc
600tgggagagcc gtcgctacag tgacctgctg ctctgggact tcctcgacgt
cccattcaac 660cagacgctca aagacctgct gctgctggcc tggctgggcc
gccactgccc caccgtgagt 720tttgtcttgc gagctcagga cgatgccttt
gtacacaccc ctgccctgct ggctcacctg 780cgggccctgc cacctgcctc
ggcccgaagc ctctacctgg gtgaggtctt tacccaggcc 840atgcctctcc
ggaagccagg aggacccttc tatgtgcccg agtccttctt cgaaggtggc
900tacccagcct atgcaagcgg gggtggctac gtcattgccg ggcgcctggc
accctggctg 960ctgcgggcgg cagcccgtgt ggcacccttc ccctttgagg
acgtctacac tggcctttgc 1020atccgagccc tgggcctggt gccccaggcc
cacccaggct tcctcacagc ctggccagca 1080gaccgcactg cggaccactg
tgctttccgc aacctgctgc tggtacggcc cctgggcccc 1140caggccagca
ttcggctctg gaaacaactg caagacccaa ggctccagtg ctga 11942397PRTHomo
sapiens 2Met Arg Cys Pro Lys Cys Leu Leu Cys Leu Ser Ala Leu Leu
Thr Leu1 5 10 15Leu Gly Leu Lys Val Tyr Ile Glu Trp Thr Ser Glu Ser
Arg Leu Ser20 25 30Lys Ala Tyr Pro Ser Pro Arg Gly Thr Pro Pro Ser
Pro Thr Pro Ala35 40 45Asn Pro Glu Pro Thr Leu Pro Ala Asn Leu Ser
Thr Arg Leu Gly Gln50 55 60Thr Ile Pro Leu Pro Phe Ala Tyr Trp Asn
Gln Gln Gln Trp Arg Leu65 70 75 80Gly Ser Leu Pro Ser Gly Asp Ser
Thr Glu Thr Gly Gly Cys Gln Ala85 90 95Trp Gly Ala Ala Ala Ala Thr
Glu Ile Pro Asp Phe Ala Ser Tyr Pro100 105 110Lys Asp Leu Arg Arg
Phe Leu Leu Ser Ala Ala Cys Arg Ser Phe Pro115 120 125Gln Trp Leu
Pro Gly Gly Gly Gly Ser Gln Val Ser Ser Cys Ser Asp130 135 140Thr
Asp Val Pro Tyr Leu Leu Leu Ala Val Lys Ser Glu Pro Gly Arg145 150
155 160Phe Ala Glu Arg Gln Ala Val Arg Glu Thr Trp Gly Ser Pro Ala
Pro165 170 175Gly Ile Arg Leu Leu Phe Leu Leu Gly Ser Pro Val Gly
Glu Ala Gly180 185 190Pro Asp Leu Asp Ser Leu Val Ala Trp Glu Ser
Arg Arg Tyr Ser Asp195 200 205Leu Leu Leu Trp Asp Phe Leu Asp Val
Pro Phe Asn Gln Thr Leu Lys210 215 220Asp Leu Leu Leu Leu Ala Trp
Leu Gly Arg His Cys Pro Thr Val Ser225 230 235 240Phe Val Leu Arg
Ala Gln Asp Asp Ala Phe Val His Thr Pro Ala Leu245 250 255Leu Ala
His Leu Arg Ala Leu Pro Pro Ala Ser Ala Arg Ser Leu Tyr260 265
270Leu Gly Glu Val Phe Thr Gln Ala Met Pro Leu Arg Lys Pro Gly
Gly275 280 285Pro Phe Tyr Val Pro Glu Ser Phe Phe Glu Gly Gly Tyr
Pro Ala Tyr290 295 300Ala Ser Gly Gly Gly Tyr Val Ile Ala Gly Arg
Leu Ala Pro Trp Leu305 310 315 320Leu Arg Ala Ala Ala Arg Val Ala
Pro Phe Pro Phe Glu Asp Val Tyr325 330 335Thr Gly Leu Cys Ile Arg
Ala Leu Gly Leu Val Pro Gln Ala His Pro340 345 350Gly Phe Leu Thr
Ala Trp Pro Ala Asp Arg Thr Ala Asp His Cys Ala355 360 365Phe Arg
Asn Leu Leu Leu Val Arg Pro Leu Gly Pro Gln Ala Ser Ile370 375
380Arg Leu Trp Lys Gln Leu Gln Asp Pro Arg Leu Gln Cys385 390
395331DNAArtificial SequenceDescription of Artificial Sequence 5'
primer for PCR 3ctcaagctta tgcgctgccc caagtgcctt c
31431DNAArtificial SequenceDescription of Artificial Sequence 3'
primer for PCR 4ctcgaattct cagcactgga gccttgggtc t
31520DNAArtificial SequenceDescription of Artificial Sequence 5'
primer for RT-PCR 5gctgttggcc gtcaagtcag 20618DNAArtificial
SequenceDescription of Artificial Sequence 3' primer for RT-PCR
6caggaagagc agccggat 18718DNAArtificial SequenceDescription of
Artificial Sequence probe for RT-PCR 7cagaacgaca ggccgtga
18829DNAArtificial SequenceDescription of Artificial Sequence 5'
primer for PCR 8gccaagctta catccgagtc ccggctcag 29929DNAArtificial
SequenceDescription of Artificial Sequence 5' primer for PCR
9gccaagctta aggcctaccc cagccctcg 291028DNAArtificial
SequenceDescription of Artificial Sequence 3' primer for PCR
10cggaattctc agcactggag ccttgggt 281155DNAArtificial
SequenceDescription of Artificial Sequence 5' primer for PCR
11ggggacaagt ttgtacaaaa aagcaggctt ccccagccct cggggcaccc cgcca
551254DNAArtificial SequenceDescription of Artificial Sequence 3'
primer for PCR 12ggggaccact ttgtacaaga aagctgggtc tcagcactgg
agccttgggt cttg 541329DNAArtificial SequenceDescription of
Artificial Sequence 5' primer for PCR 13gccaagctta catccgagtc
ccggctcag 291429DNAArtificial SequenceDescription of Artificial
Sequence 5' primer for PCR 14gccaagctta aggcctaccc cagccctcg
291528DNAArtificial SequenceDescription of Artificial Sequence 3'
primer for PCR 15cggaattctc agcactggag ccttgggt 2816372PRTHomo
sapiens 16Thr Ser Glu Ser Arg Leu Ser Lys Ala Tyr Pro Ser Pro Arg
Gly Thr1 5 10 15Pro Pro Ser Pro Thr Pro Ala Asn Pro Glu Pro Thr Leu
Pro Ala Asn20 25 30Leu Ser Thr Arg Leu Gly Gln Thr Ile Pro Leu Pro
Phe Ala Tyr Trp35 40 45Asn Gln Gln Gln Trp Arg Leu Gly Ser Leu Pro
Ser Gly Asp Ser Thr50 55 60Glu Thr Gly Gly Cys Gln Ala Trp Gly Ala
Ala Ala Ala Thr Glu Ile65 70 75 80Pro Asp Phe Ala Ser Tyr Pro Lys
Asp Leu Arg Arg Phe Leu Leu Ser85 90 95Ala Ala Cys Arg Ser Phe Pro
Gln Trp Leu Pro Gly Gly Gly Gly Ser100 105 110Gln Val Ser Ser Cys
Ser Asp Thr Asp Val Pro Tyr Leu Leu Leu Ala115 120 125Val Lys Ser
Glu Pro Gly Arg Phe Ala Glu Arg Gln Ala Val Arg Glu130 135 140Thr
Trp Gly Ser Pro Ala Pro Gly Ile Arg Leu Leu Phe Leu Leu Gly145 150
155 160Ser Pro Val Gly Glu Ala Gly Pro Asp Leu Asp Ser Leu Val Ala
Trp165 170 175Glu Ser Arg Arg Tyr Ser Asp Leu Leu Leu Trp Asp Phe
Leu Asp Val180 185 190Pro Phe Asn Gln Thr Leu Lys Asp Leu Leu Leu
Leu Ala Trp Leu Gly195 200 205Arg His Cys Pro Thr Val Ser Phe Val
Leu Arg Ala Gln Asp Asp Ala210 215 220Phe Val His Thr Pro Ala Leu
Leu Ala His Leu Arg Ala Leu Pro Pro225 230 235 240Ala Ser Ala Arg
Ser Leu Tyr Leu Gly Glu Val Phe Thr Gln Ala Met245 250 255Pro Leu
Arg Lys Pro Gly Gly Pro Phe Tyr Val Pro Glu Ser Phe Phe260 265
270Glu Gly Gly Tyr Pro Ala Tyr Ala Ser Gly Gly Gly Tyr Val Ile
Ala275 280 285Gly Arg Leu Ala Pro Trp Leu Leu Arg Ala Ala Ala Arg
Val Ala Pro290 295 300Phe Pro Phe Glu Asp Val Tyr Thr Gly Leu Cys
Ile Arg Ala Leu Gly305 310 315 320Leu Val Pro Gln Ala His Pro Gly
Phe Leu Thr Ala Trp Pro Ala Asp325 330 335Arg Thr Ala Asp His Cys
Ala Phe Arg Asn Leu Leu Leu Val Arg Pro340 345 350Leu Gly Pro Gln
Ala Ser Ile Arg Leu Trp Lys Gln Leu Gln Asp Pro355 360 365Arg Leu
Gln Cys37017282PRTHomo sapiens 17Arg Arg Phe Leu Leu Ser Ala Ala
Cys Arg Ser Phe Pro Gln Trp Leu1 5 10 15Pro Gly Gly Gly Gly Ser Gln
Val Ser Ser Cys Ser Asp Thr Asp Val20 25 30Pro Tyr Leu Leu Leu Ala
Val Lys Ser Glu Pro Gly Arg Phe Ala Glu35 40 45Arg Gln Ala Val Arg
Glu Thr Trp Gly Ser Pro Ala Pro Gly Ile Arg50 55 60Leu Leu Phe Leu
Leu Gly Ser Pro Val Gly Glu Ala Gly Pro Asp Leu65 70 75 80Asp Ser
Leu Val Ala Trp Glu Ser Arg Arg Tyr Ser Asp Leu Leu Leu85 90 95Trp
Asp Phe Leu Asp Val Pro Phe Asn Gln Thr Leu Lys Asp Leu Leu100 105
110Leu Leu Ala Trp Leu Gly Arg His Cys Pro Thr Val Ser Phe Val
Leu115 120 125Arg Ala Gln Asp Asp Ala Phe Val His Thr Pro Ala Leu
Leu Ala His130 135 140Leu Arg Ala Leu Pro Pro Ala Ser Ala Arg Ser
Leu Tyr Leu Gly Glu145 150 155 160Val Phe Thr Gln Ala Met Pro Leu
Arg Lys Pro Gly Gly Pro Phe Tyr165 170 175Val Pro Glu Ser Phe Phe
Glu Gly Gly Tyr Pro Ala Tyr Ala Ser Gly180 185 190Gly Gly Tyr Val
Ile Ala Gly Arg Leu Ala Pro Trp Leu Leu Arg Ala195 200 205Ala Ala
Arg Val Ala Pro Phe Pro Phe Glu Asp Val Tyr Thr Gly Leu210 215
220Cys Ile Arg Ala Leu Gly Leu Val Pro Gln Ala His Pro Gly Phe
Leu225 230 235 240Thr Ala Trp Pro Ala Asp Arg Thr Ala Asp His Cys
Ala Phe Arg Asn245 250 255Leu Leu Leu Val Arg Pro Leu Gly Pro Gln
Ala Ser Ile Arg Leu Trp260 265 270Lys Gln Leu Gln Asp Pro Arg Leu
Gln Cys275 280181845DNAMouse 18ggaggacgca cagctgcgag gagggagtcc
gggcagggct ttacccgagg acccccagag 60ctggcggaag ctggacccag aggtacctgg
ggccccaggc cctggggtgg ggttactgga 120ggaggtaggt aggcttccaa
gaaggtaaaa aggagtttcc ccgggaagct gggactcctg 180aagagacaga
ggaatgaggg aaggggagta ggaagagccg ttggagcgat actgcaaata
240gatataacac catgactgca gaaaaggaaa gaatgggggg tcgaggggag
gcggtgttca 300gtctaggata acgttaagtt gggtactgta gttcagtctg
cctagggtca gagtctcaga 360agccattaac agaactgggt aggacctagg
ctgcttgcct gggcttcgct gggccgtctt 420tggagatcca ccctgcaccc
taaagacttc tgtggctcct tgtgactctt gcagccccac 480tggtggccct
ttccctgggc cgggtcatgc gttgccgcaa gtgccagctc tgcctgtcag
540cactgctcac actcctgggc ctcaaagtat acatcgagtg gacatccgag
tcctggctta 600aaaaggctga accccggggc gctctgccca gtcccacacc
acccaatgct gagcccactc 660tgcccaccaa cctctcagca cgcctgggtc
agactggccc actgtcctct gcttactgga 720accagcagca gcggcagctg
ggagtcctgc ccagtacgga ctgtcagact tgggggactg 780ttgctgcctc
ggagatcttg gacttcatcc tgtaccccca ggagcttcgg cgcttcttgc
840tgtcggcggc ctgtaggagc tttccactat ggctgcctgc aggagaaggc
agccctgtgg 900ccagctgctc tgataaggat gtaccctact tgctactggc
tgtcaaatca gaaccaggac 960actttgcagc acggcaggct gtgagggaga
cctggggcag cccagttgct gggacccggt 1020tgctcttcct gctggggtcc
cccctaggaa tgggggggcc tgacttaaga tcactggtga 1080cgtgggaaag
ccggcgctat ggtgacctac tgctctggga cttcctggat gttccctaca
1140accggacact caaggacctg ctgctgctga cctggctgag ccaccactgc
cccgatgtca 1200attttgtcct gcaggttcag gatgatgcct ttgtgcacat
cccagcccta ctggagcacc 1260tgcagactct gccacccacc tgggcccgca
gcctctacct gggtgagatc ttcacccagg 1320ccaaaccgct ccgcaagccc
ggaggaccct tctatgtgcc gaagaccttc tttgaagggg 1380actatccagc
ctatgcgagt ggaggtggct atgtaatctc aggacgcctg gcaccctggc
1440tgctgcaggc ggcagctcgc gtggcaccct tcccctttga tgatgtctac
actggcttct 1500gcttccgtgc cctgggctta gcaccccgtg cccatccagg
cttcctcaca gcctggccag 1560cagaacgtac cagggacccc tgcgccgtgc
gaggcctgct cttggtgcat ccagtcagcc 1620ctcaggacac catttggctc
tggagacatc tgtgggtccc agagctccag tgctgaccgg 1680cagagacaag
ctggggtggg tgggtgctga cctggcctga gtctctccta gagacaagct
1740ggggtgggtg gggctgacct ggcctgagtc tctcctaaac ccttcttagc
caaggtggca 1800gactgtgttt atctacttta tggttttgaa aaatgtgtcc ttcct
1845191170DNAMouse 19atgcgttgcc gcaagtgcca gctctgcctg tcagcactgc
tcacactcct gggcctcaaa 60gtatacatcg agtggacatc cgagtcctgg cttaaaaagg
ctgaaccccg gggcgctctg 120cccagtccca caccacccaa tgctgagccc
actctgccca ccaacctctc agcacgcctg 180ggtcagactg gcccactgtc
ctctgcttac tggaaccagc agcagcggca gctgggagtc 240ctgcccagta
cggactgtca gacttggggg actgttgctg cctcggagat cttggacttc
300atcctgtacc cccaggagct tcggcgcttc ttgctgtcgg cggcctgtag
gagctttcca 360ctatggctgc ctgcaggaga aggcagccct gtggccagct
gctctgataa ggatgtaccc 420tacttgctac tggctgtcaa atcagaacca
ggacactttg cagcacggca ggctgtgagg 480gagacctggg gcagcccagt
tgctgggacc cggttgctct tcctgctggg gtccccccta 540ggaatggggg
ggcctgactt aagatcactg gtgacgtggg aaagccggcg ctatggtgac
600ctactgctct gggacttcct ggatgttccc tacaaccgga cactcaagga
cctgctgctg 660ctgacctggc tgagccacca ctgccccgat gtcaattttg
tcctgcaggt tcaggatgat 720gcctttgtgc acatcccagc cctactggag
cacctgcaga ctctgccacc cacctgggcc 780cgcagcctct acctgggtga
gatcttcacc caggccaaac cgctccgcaa gcccggagga 840cccttctatg
tgccgaagac cttctttgaa ggggactatc cagcctatgc gagtggaggt
900ggctatgtaa tctcaggacg cctggcaccc tggctgctgc aggcggcagc
tcgcgtggca 960cccttcccct ttgatgatgt ctacactggc ttctgcttcc
gtgccctggg cttagcaccc 1020cgtgcccatc caggcttcct cacagcctgg
ccagcagaac gtaccaggga cccctgcgcc 1080gtgcgaggcc tgctcttggt
gcatccagtc agccctcagg acaccatttg gctctggaga 1140catctgtggg
tcccagagct ccagtgctga 117020389PRTMouse 20Met Arg Cys Arg Lys Cys
Gln Leu Cys Leu Ser Ala Leu Leu Thr Leu1 5 10 15Leu Gly Leu Lys Val
Tyr Ile Glu Trp Thr Ser Glu Ser Trp Leu Lys20 25 30Lys Ala Glu Pro
Arg Gly Ala Leu Pro Ser Pro Thr Pro Pro Asn Ala35 40 45Glu Pro Thr
Leu Pro Thr Asn Leu Ser Ala Arg Leu Gly Gln Thr Gly50 55 60Pro Leu
Ser Ser Ala Tyr Trp Asn Gln Gln Gln Arg Gln Leu Gly Val65 70 75
80Leu Pro Ser Thr Asp Cys Gln Thr Trp Gly Thr Val Ala Ala Ser Glu85
90 95Ile Leu Asp Phe Ile Leu Tyr Pro Gln Glu Leu Arg Arg Phe Leu
Leu100 105 110Ser Ala Ala Cys Arg Ser Phe Pro Leu Trp Leu Pro Ala
Gly Glu Gly115 120 125Ser Pro Val Ala Ser Cys Ser Asp Lys Asp Val
Pro Tyr Leu Leu Leu130 135 140Ala Val Lys Ser Glu Pro Gly His Phe
Ala Ala Arg Gln Ala Val Arg145 150 155 160Glu Thr Trp Gly Ser Pro
Val Ala Gly Thr Arg Leu Leu Phe Leu Leu165 170 175Gly Ser Pro Leu
Gly Met Gly Gly Pro Asp Leu Arg Ser Leu Val Thr180 185 190Trp Glu
Ser Arg Arg Tyr Gly Asp Leu Leu Leu Trp Asp Phe Leu Asp195 200
205Val Pro Tyr Asn Arg Thr Leu Lys Asp Leu Leu Leu Leu Thr Trp
Leu210 215 220Ser His His Cys Pro Asp Val Asn Phe Val Leu Gln Val
Gln Asp Asp225 230 235 240Ala Phe Val His Ile Pro Ala Leu Leu Glu
His Leu Gln Thr Leu Pro245 250 255Pro Thr Trp Ala Arg Ser Leu Tyr
Leu Gly Glu Ile Phe Thr Gln Ala260 265 270Lys Pro Leu Arg Lys Pro
Gly Gly Pro Phe Tyr Val Pro Lys Thr Phe275 280 285Phe Glu Gly Asp
Tyr Pro Ala Tyr Ala Ser Gly Gly Gly Tyr Val Ile290 295 300Ser Gly
Arg Leu Ala Pro Trp Leu Leu Gln Ala Ala Ala Arg Val Ala305 310 315
320Pro Phe Pro Phe Asp Asp Val Tyr Thr Gly Phe Cys Phe Arg Ala
Leu325 330 335Gly Leu Ala Pro Arg Ala His Pro Gly Phe Leu Thr Ala
Trp Pro Ala340 345 350Glu Arg Thr Arg Asp Pro Cys Ala Val Arg Gly
Leu Leu Leu Val His355 360 365Pro Val Ser Pro Gln Asp Thr Ile Trp
Leu Trp Arg His Leu Trp Val370 375 380Pro Glu Leu Gln Cys385
* * * * *