U.S. patent application number 10/212933 was filed with the patent office on 2003-01-09 for polypeptide of n-acetylglucosamine-6-o-sulfotransferase and dna encoding the same.
Invention is credited to Habuchi, Osami, Kadomatsu, Kenji, Kannagi, Reiji, Muramatsu, Hideki, Muramatsu, Takashi, Uchimura, Kenji.
Application Number | 20030008366 10/212933 |
Document ID | / |
Family ID | 26394743 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030008366 |
Kind Code |
A1 |
Uchimura, Kenji ; et
al. |
January 9, 2003 |
Polypeptide of N-acetylglucosamine-6-O-sulfotransferase and DNA
encoding the same
Abstract
Apolypeptide of N-acetylglucosamine-6-O-sulfotransferase and a
DNA encoding the peptide are provided. The polypeptide is (a) or
(b) below: (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 2; or (b) a polypeptide which comprises
an amino acid sequence including substitution, deletion, insertion
or transposition of one or few amino acids in the amino acid
sequence of (a) and which has an enzymatic activity to transfer a
sulfate group from a sulfate group donor to a hydroxyl group at 6
position of an N-acetylglucosamine residue located at a
non-reducing end of an oligosaccharide represented by the formula
I: GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I) wherein GlcNAc represents
an N-acetylglucosamine residue, Gal represents a galactose residue,
.beta.1-3 represents a .beta.1-3 glycosidic linkage, and .beta.1-4
represents a .beta.1-4 glycosidic linkage.
Inventors: |
Uchimura, Kenji;
(Nagoya-shi, JP) ; Muramatsu, Hideki;
(Shizuoka-ken, JP) ; Kadomatsu, Kenji;
(Nagoya-shi, JP) ; Kannagi, Reiji; (Nagoya-shi,
JP) ; Habuchi, Osami; (Nagoya-shi, JP) ;
Muramatsu, Takashi; (Nagoya-shi, JP) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
26394743 |
Appl. No.: |
10/212933 |
Filed: |
August 5, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10212933 |
Aug 5, 2002 |
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09471867 |
Dec 23, 1999 |
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6455289 |
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09471867 |
Dec 23, 1999 |
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09263023 |
Mar 5, 1999 |
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6037159 |
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Current U.S.
Class: |
435/183 ;
514/19.1; 514/20.9 |
Current CPC
Class: |
C07K 16/2896 20130101;
C12P 19/04 20130101; C07K 16/44 20130101; C12N 9/13 20130101 |
Class at
Publication: |
435/183 ;
514/12 |
International
Class: |
C12N 009/00; A61K
038/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 5, 1998 |
JP |
10-54007 |
Jun 24, 1998 |
JP |
10-177844 |
Claims
What is claimed is:
1. An isolated polypeptide having an amino acid sequence shown as
SEQ ID No. 2 or a variant thereof wherein the polypeptide has an
enzymatic activity to transfer a sulfate group from a sulfate group
donor to a hydroxyl group at 6 position of an N-acetylglucosamine
residue located at a non-reducing end of an oligosaccharide
represented by formula I: GlcNAc.beta.11-3Gal.beta.1-4GlcNAc (I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal
represents a galactose residue, .beta.1-3 represents a .beta.1-3
glycosidic linkage, and .beta.1-4 represents a .beta.1-4 glycosidic
linkage.
2. An isolated polypeptide having an amino acid sequence shown as
SEQ ID No. 4 or a variant thereof wherein the polypeptide has an
enzymatic activity to transfer a sulfate group from a sulfate group
donor to a hydroxyl group at 6 position of an N-acetylglucosamine
residue located at a non-reducing end of an oligosaccharide
represented by formula I: GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal
represents a galactose residue, .beta.1-3 represents a .beta.1-3
glycosidic linkage, and .beta.1-4 represents a .beta.1-4 glycosidic
linkage.
3. An isolated polypeptide having the following properties: (a)
Action: a sulfate group is transferred from a sulfate group donor
to a hydroxyl group at 6 position of an N-acetylglucosamine residue
located at a non-reducing end of an oligosaccharide represented by
the formula I: GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I) wherein GlcNAc
represents an N-acetylglucosamine residue, Gal represents a
galactose residue, .beta.1-3 represents a .beta.1-3 glycosidic
linkage, and .beta.1-4 represents a .beta.1-4 glycosidic linkage.
(b) Substrate specificity: a sulfate group is not transferred to
any of the substances selected from the group consisting of;
chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan
sulfate, keratan sulfate, desulfated keratan sulfate,
CDSNS-heparin, mucin from porcine stomach, mucin from bovine
submaxillary gland, and an oligosaccharide represented by the
formula II: Gal.beta.1-4 GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (II)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal
represents a galactose residue, .beta.1-3 represents a .beta.1-3
glycosidic linkage, and .beta.1-4 represents a .beta.1-4 glycosidic
linkage; and (c) the N-terminal amino acid sequence comprises an
amino acid sequence represented by amino acid numbers 1 to 48 in
SEQ ID No: 2.
4. A method of producing a sulfated sugar represented by the
formula III: (SO.sub.4-6)GlcNAc-R (III) wherein GlcNAc represents
an N-acetylglucosamine residue; SO.sub.4-6 means that a hydroxyl
group at position 6 is sulfated, and --R represents a hydrogen atom
or a sugar residue bonded by a glycosidic linkage, said method
comprising; reacting the polypeptide described in claim 1 with a
sugar chain represented by the formula IV: GlcNAc-R (IV) wherein
GlcNAc represents an N-acetylglucosamine residue, and --R
represents a hydrogen atom or a sugar residue bonded by a
glycosidic linkage.
5. An isolated antibody that reacts with 6-sulfated sialyl Lewis X
but does not react with any of the substances selected from the
group consisting of; 6'-sulfated sialyl Lewis X, 6,6-bis-sulfated
sialyl Lewis X, 6-sulfated Lewis X and Lewis X.
6. An isolated antibody that reacts with a sugar chain represented
by formula V: Gal.beta.1-4(Fuc.alpha.1-3)(SO.sub.4-6)GlcNAc (V)
7. An antibody that reacts with a sugar chain represented by
formula VI: NeuAc.alpha.2-3Gal.beta.1-4(SO.sub.4-6)GlcNAc (VI)
8. A method of producing a sulfated sugar represented by the
formula III: (SO.sub.4-6)GlcNAc-R (III) wherein GIcNAc represents
an N-acetylglucosamine residue; SO.sub.4-6 means that a hydroxyl
group at position 6 is sulfated, and --R represents a hydrogen atom
or a sugar residue bonded by a glycosidic linkage, said method
comprising; reacting the polypeptide described in claim 2 with a
sugar chain represented by the formula IV: GlcNAc-R (IV) wherein
GlcNAc represents an N-acetylglucosamine residue, and --R
represents a hydrogen atom or a sugar residue bonded by a
glycosidic linkage.
9. A method of producing a sulfated sugar represented by the
formula III: (SO.sub.4-6)GlcNAc-R (III) wherein GlcNAc represents
an N-acetylglucosamine residue; SO.sub.4-6 means that a hydroxyl
group at position 6 is sulfated, and --R represents a hydrogen atom
or a sugar residue bonded by a glycosidic linkage, said method
comprising; reacting the polypeptide described in claim 3 with a
sugar chain represented by the formula IV: GlcNAc-R (IV) Wherein
GlcNAc represents an N-acetylglucosamine residue, and --R
represents a hydrogen atom or a sugar residue bonded by a
glycosidic linkage.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a polypeptide of
N-acetylglucosamine-6-O-sulfotransferase and a DNA having a
nucleotide sequence encoding the polypeptide.
DESCRIPTION OF THE PRIOR ART
[0002] The closest prior art is described below.
[0003] It has been described in Biochem. J., 319, 209-216 (1996)
and J. Biol. Chem. 272, 29493-29501 (1997) that rat and human
microsome fractions had an N-acetylglucosamine-6-O-sulfotransferase
activity. However, there has been so far no report about isolation
and identification of a polypeptide of
N-acetylglucosamine-6-O-sulfotransfera- se. A DNA encoding this
polypeptide has not also been known.
[0004] Once a polypeptide of
N-acetylglucosamine-6-O-sulfotransferase is obtained, it can be
used for synthesis of sugar chains such as GlyCAM-1 that is a
ligand of L-selectin (which is involved in homing of lymphocytes
and rolling of leukocytes occurring at the early stage of
inflammation). A DNA encoding this polypeptide would be expected to
be used for large scale production of the polypeptide, or
artificial synthesis of GlyCAM-1 (having a structure of
NeuAc.alpha.2-3Gal.beta.1-4(- Fuc.alpha.1-3) (SO.sub.4-6)GlcNAc-)
by using transformants which harbors the DNA.
SUMMARY OF THE INVENTION
[0005] An object of the present invention is to provide a
polypeptide of N-acetylglucosamine-6-O-sulfotransferase and a DNA
encoding the polypeptide.
[0006] The present inventors have succeeded in cloning a DNA
encoding a polypeptide of N-acetylglucosamine-6-O-sulfotransferase,
having an activity to specifically transfer a sulfate group to a
hydroxyl group at 6 position of an N-acetylglucosamine residue
located at a non-reducing end of GlcNAc.beta.1-3Gal.beta.1-4GlcNAc,
wherein GlcNAc represents an N-acetylglucosamine-residue, Gal
represents a galactose residue, .beta.1-3 represents a .beta.1-3
glycosidic linkage, and .beta.1-4 represents a .beta.1-4 glycosidic
linkage. The inventors have also confirmed that the polypeptide of
N-acetylglucosamine-6-O-sulfotransferas- e was expressed by the DNA
and identified the polypeptide, thereby completing the present
invention.
[0007] The present invention provides a polypeptide of (a) or (b)
below (hereinafter sometimes referred to as "the polypeptide of the
present invention"):
[0008] (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 2; or
[0009] (b) a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a) and which has
an enzymatic activity to transfer a sulfate group from a sulfate
group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end of an
oligosaccharide represented by the formula I:
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I)
[0010] wherein GlcNAc represents an N-acetylglucosamine residue,
Gal represents a galactose residue, .beta.1-3 represents a
.beta.1-3 glycosidic linkage, and .beta.1-4 represents a .beta.1-4
glycosidic linkage.
[0011] The polypeptide of the present invention also includes a
polypeptide of (a) or (b) below:
[0012] (a) a polypeptide consisting of the amino acid sequence
represented by SEQ ID NO: 4; or
[0013] (b) a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a) and which has
an enzymatic activity to transfer a sulfate group from a sulfate
group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end of an
oligosaccharide represented by the formula I:
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I)
[0014] wherein GlcNAc represents an N-acetylglucosamine residue,
Gal represents a galactose residue, .beta.1-3 represents a
.beta.1-3 glycosidic linkage, and .beta.1-4 represents a .beta.1-4
glycosidic linkage.
[0015] The polypeptide of the present invention also includes a
polypeptide having the following properties:
[0016] (1) Action: a sulfate group is transferred from a sulfate
group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end of an
oligosaccharide represented by the formula I:
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I)
[0017] wherein GlcNAc represents an N-acetylglucosamine residue,
Gal represents a galactose residue, .beta.1-3 represents a
.beta.1-3 glycosidic linkage, and .beta.1-4 represents a .beta.1-4
glycosidic linkage;
[0018] (2) Substrate specificity: sulfate group is not transferred
to chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate,
dermatan sulfate, keratan sulfate, desulfated keratan sulfate,
CDSNS-heparin, mucin from porcine stomach, mucin from bovine
submaxillary gland, and an oligosaccharide represented by the
formula II:
Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (II)
[0019] wherein GlcNAc represents an N-acetylglucosamine residue,
Gal represents a galactose residue, .beta.1-3 represents a
.beta.1-3 glycosidic linkage, and .beta.1-4 represents a .beta.1-4
glycosidic linkage; and
[0020] (3) N-terminal amino acid sequence comprises an amino acid
sequence represented by amino acid numbers 1 to 48 in SEQ ID NO:
2.
[0021] The present invention provides a DNA encoding a polypeptide
of (a) or (b) below (hereinafter sometimes referred to as "the DNA
of the present invention"):
[0022] (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 2; or
[0023] (b) a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a) and which has
an enzymatic activity to transfer a sulfate group from a sulfate
group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end of an
oligosaccharide represented by the formula I:
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I)
[0024] wherein GlcNAc represents an N-acetylglucosamine residue,
Gal represents a galactose residue, .beta.1-3 represents a
.beta.1-3 glycosidic linkage, and .beta.1-4 represents a .beta.1-4
glycosidic linkage.
[0025] The DNA of the present invention also includes a DNA
encoding a polypeptide of (a) or (b) below:
[0026] (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 4; or
[0027] (b) a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a) and which has
an enzymatic activity to transfer a sulfate group from a sulfate
group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end of an
oligosaccharide represented by the formula I:
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I)
[0028] wherein GlcNAc represents an N-acetylglucosamine residue,
Gal represents a galactose residue, .beta.1-3 represents a
.beta.1-3 glycosidic linkage, and .beta.1-4 represents a .beta.1-4
glycosidic linkage.
[0029] The DNA of the present invention is preferably a DNA
comprising a nucleotide sequence represented by nucleotide numbers
470 to 1918 in SEQ ID NO: 1 and a DNA comprising a nucleotide
sequence represented by nucleotide numbers 390 to 1841 in SEQ ID
NO: 3.
[0030] The present invention also provides a method of producing a
sulfated sugar (hereinafter sometimes referred to as "the producing
method 1 of the present invention") represented by the formula
III:
(SO.sub.4-6)GlcNAc-R (III)
[0031] wherein GlcNAc represents an N-acetylglucosamine residue;
SO.sub.4-6 means that a hydroxyl group at 6 position is sulfated,
and --R represents a hydrogen atom or a sugar residue bonded by a
glycosidic linkage, which comprises a step of reacting the
above-described polypeptide with a sugar chain represented by the
formula IV:
GlcNAc-R (IV)
[0032] wherein GlcNAc represents an N-acetylglucosamine residue,
and --R represents a hydrogen atom or a sugar residue bonded by a
glycosidic linkage.
[0033] Furthermore, the present invention provides a method of
producing a sulfated sugar (hereinafter sometimes referred to as
"the producing method 2 of the present invention"), which comprises
a step of transfecting a cell with the DNA of the present invention
into and then culturing the cell. A preferred embodiment of this
method is a method of transfecting a cell with the DNA of the
present invention and a cDNA encoding fucosyltransferase
concurrently.
[0034] The present invention further provides an antibody that
reacts with 6-sulfated sialyl Lewis X but does not react with
6'-sulfated sialyl Lewis X, 6,6'-bis-sulfated sialyl Lewis X,
6-sulfated Lewis X and Lewis X.
[0035] The present invention still further provides an antibody
that specifically reacts with a sugar chain represented by the
following formula:
Gal.beta.1-4(Fuc.alpha.1-3)(SO.sub.4-6)GlcNAc
[0036] The present invention also provides an antibody that reacts
with a sugar chain represented by the following formula:
NeuAc.alpha.2-3Gal.beta.1-4(SO.sub.4-6)GlcNAc.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 shows the hydropathy plot of the polypeptide of the
present invention derived from mouse. The hydropathy plot was
calculated by the method of Kyte and Doolittle (J. Mol. Biol., 157,
105-132 (1982)) with a window of 11 amino acids.
[0038] FIG. 2 shows the substrate specificity of the DNA of the
present invention derived from mouse.
[0039] A:GlcNAc.beta.1-3Gal.beta.1-4GlcNAc as the sulfate group
acceptor.
[0040] B: Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (L1L1) as
the sulfate group acceptor. After the enzyme reaction, the products
were analyzed by Superdex 30 Gel chromatography. ".circle-solid."
indicates in the presence of acceptors; ".smallcircle." indicates
in the absence of acceptors. Arrows indicates the elution position
of acceptors.
[0041] FIG. 3 shows the assay result of sulfated products with the
polypeptide of the present invention derived from mouse.
[0042] A: .sup.35S-Labeled GlcNAc.beta.1-3Gal.beta.1-4GlcNAc was
N-deacetylated, deamino-cleaved and reduced and separated by paper
chromatography. Arrows 1,2, and 3 indicate the migration position
of standard substances: 1,
(SO.sub.4-6)Gal.beta.1-4(SO.sub.4-6)2,5-anhydroma- nnitol; 2,
Gal.beta.1-4(SO4-6) 2,5-anhydromannitol; 3,
(SO4-6)2,5-anhydromannitol.
[0043] B: .sup.35S Labeled (SO.sub.4-6).sub.2, 5-anhydromannitol
fraction, which was shown by a horizontal bar in A) was mixed with
.sup.3H-labeled (SO.sub.4-6)2,5-anhydromannitol (standard) and
analyzed by HPLC using a SAX-10 column. Radioactivity of .sup.3H
(.smallcircle.) and .sup.35S (.circle-solid.) of each fracton was
determined.
[0044] FIG. 4 shows the assay result of the specificity of AG223
and G72 monoclonal antibodies by ELISA. Glycolipids were
immobilized at the bottom of 96-well culture plates and ELISA
assays were performed. "S" indicates NeuAc, "Lex" indicates Lewis X
ceramide, "PG" indicates paragloboside, "SL2L4" indicates
NeuAc.alpha.2-3Gal.beta.1-4(SO.sub.4-6)G- lcNAc.beta.1-3
(SO.sub.4-6) Gal.beta.1-4 (SO.sub.4-6) GlcNAc-chol esteryl aniline,
respectively.
[0045] FIG. 5 shows the expression of G72 antigen by transfection
with the DNA of the present invention derived from mouse.
[0046] COS-7 cells before transfection (A, D), or the cells
transfected with the sense (the correct orientation) cDNA (B, E) or
with the antisense (the reverse orientation) cDNA (C, F) were
reacted with G72 monoclonal antibody and analyzed by FACS. Thick
lines are the pattern after reaction with the antibody, and thin
lines are those before the reaction. "-NANase" indicates a result
before neuramimidase digestion, "+NANase" indicates a result after
digestion with 0.02 units/ml of Arthrobacter ureafaciens
neuramimidase in PBS, (pH 7.4) at 37.degree. C. for 30 min.
[0047] FIG. 6 shows the expression of AG223 (6-sulfo Lewis X)
antigen by double transfection with the DNA of the present
invention derived from mouse and fucosyltransferase IV cDNA. COS-7
cells transfected with the fucosyltransferase cDNA alone (A, D),
the DNA of the present invention alone (B, E) or both the DNA (C,
F) were reacted with AG223 monoclonal antibody (A-C) or with LeuMl
(anti-Lewis X antibody) (D-F), and analyzed by FACS. Thick lines
are the pattern after reaction with antibody, and thin lines are
those before the reaction.
[0048] FIG. 7 shows the expression of G72 antigen (6-sulfo sialyl
N-acetyllactosamine antigen) by transfection with the DNA of the
present invention derived from human, and the expression of G152
antigen (6-sulfo sialyl Lewis X antigen) by double transfection
with the DNA of the present invention derived from human and
fucosyltransferase IV cDNA.
[0049] A data shows mean fluorescence intensity of antigen positive
group in transfected cells.
[0050] FIG. 8 shows the result of Northern and Southern analyses of
the DNA of the present invention derived from mouse.
[0051] A: Nothern blot analysis. Arrowheads indicate the positions
of different mRNAs of 13.5, 9.8, and 3.9 kb. The positions of
ribosomal RNAs are indicated at the left. Hybridization with a
glyceroaldehyde 3-phosphate dehydrogenase probe revealed similar
intensity of bands in each lane, except that in the pancreas the
intensity was weak. B: Genomic Southern blot analysis. Single bands
were detected after digestion with EcoRI, EcoRV or SacI.
DETAILED DESCRIPTION OF THE INVENTION
[0052] The mode for carrying out the present invention is described
below.
[0053] Unless otherwise specified herein, Gal represents a
galactose residue, GlcNAc represents an N-acetylglucosamine
residue, NeuAc represents an N-acetylneuraminic acid residue, Fuc
represents a fucose residue, .beta.1-3 represents a .beta.1-3
glycosidic linkage, .beta.1-4 represents a .beta.1-4 glycosidic
linkage, .alpha.2-3 represents a .alpha.2-3 glycosidic linkage,
.alpha.1-3 represents a .beta.1-3 glycosidic linkage, SO.sub.4-6
represents that a hydroxyl group at 6 position is sulfated, and Cer
represents a ceramide residue, respectively.
[0054] <1> The Polypeptides of the Present Invention
[0055] The polypeptide of the present invention is a polypeptide of
(a) or (b) below:
[0056] (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 2; or
[0057] (b) a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a) and which has
an enzymatic activity to transfer a sulfate group from a sulfate
group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end of an
oligosaccharide represented by the formula I:
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I).
[0058] The polypeptide (a) is preferred among them.
[0059] In this specification, the term "a polypeptide which
comprises an amino acid sequence including substitution, deletion,
insertion or transposition of one or few amino acids in the amino
acid sequence of (a) and which has an enzymatic activity to
transfer a sulfate group from a sulfate group donor to a hydroxyl
group at 6 position of an N-acetylglucosamine residue located at a
non-reducing end of an oligosaccharide represented by the formula I
(GlcNAc.beta.1-3Gal.beta.1-4- GlcNAc)" means that one or more amino
acid residues of the polypeptide may be substituted, deleted,
inserted, or transferred as long as such modification does not
substantially affect the activity to selectively transfer a sulfate
group from a sulfate group donor to a hydroxyl group at 6 position
of an N-acetylglucosamine residue located at a non-reducing end of
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc.
[0060] Mutation such as substitution, deletion, insertion, or
transposition of amino acid residues may occur in the amino acid
sequence of the polypeptides existing in nature due to, for
example, the modifying reaction of the biosynthesized polypeptides
in the living oraganisms or during their purification as well as
polymorphism and mutation of the DNAs encoding the polypeptides,
nevertheless some of mutated polypeptides are known to have
substantially the same physiological and biological activities as
the intact polypeptides that have not been mutated. The polypeptide
of the present invention includes those having slightly different
structures but not having a significant difference in the
functions. The polypeptide of the present invention also includes
those which have been artificially treated to have mutation as
described above in the amino acid sequences. In this case, a
further variety of mutants can be produced. For example, a
polypeptide having a human interleukin 2 (IL-2) amino acid
sequence, in which a cysteine residue has been replaced with a
serine residue, is known to retain the interleukin 2 activities
(Science 224, 1431 (1984)). Furthermore, a polypeptide of certain
kind is known to have a peptide region that is not essential for
exhibiting its activities. Examples of such polypeptides include a
signal peptide contained in a polypeptide that is secreted
extracellularly and a pro-sequence found in a precursor of
protease, and the like. Most of these regions are removed after
translation or upon conversion into an active form of the
polypeptides. These polypeptides exist in different primary
structures but finally have equivalent functions. Such polypeptides
are also included in the polypeptide of the present invention.
[0061] The term "few amino acids" used herein means the number of
amino acid residues that may be mutated to the extent that the
activities of the polypeptide of the present invention are not
lost. For example, in a polypeptide consisting of 400 amino acid
residues, about 20 or less of amino acid residues may be
mutated.
[0062] The enzymatic activity to transfer a sulfate group from a
sulfate group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end of an
oligosaccharide represented by GlcNAc.beta.1-3Gal.beta.1-4GlcNAc
can be determined by the method of measuring the activities of the
polypeptide of the present invention as will be described later.
Determining whether the presence or absence of the activities of
the polypeptide of the present invention as an index, one of
ordinary skilled in the art would readily select substitution,
deletion, insertion, or transposition of one or more amino acid
residues, which does not substantially affect the activities of the
polypeptide.
[0063] This polypeptide has been obtained from a mouse originally.
As a matter of course, however, the polypeptide of the present
invention also includes polypeptides produced by genetic
engineering procedure or chemical synthesis.
[0064] The polypeptide of the present invention also includes a
polypeptide of (a) or (b) below:
[0065] (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 4; or
[0066] (b) a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a) and which has
an enzymatic activity to transfer a sulfate group from a sulfate
group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end of an
oligosaccharide represented by the formula I:
[0067] GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I)
[0068] Among the above polypeptides, the polypeptide (a) (the
polypeptide consisting of an amino acid sequence represented by SEQ
ID NO: 4) is preferred because it has 85% or more homology to the
above-described polypeptide (a) of the present invention (the
polypeptide consisting of an amino acid sequence represented by SEQ
ID NO: 2). Thus, the polypeptide of the present invention includes
polypeptides having 85% or more homology to the polypeptide
consisting of the amino acid sequence represented by SEQ ID NO:
2).
[0069] This polypeptide has been derived from human originally. As
a matter of course, however, the polypeptide of the present
invention also includes polypeptides produced by genetic
engineering procedure or chemical synthesis.
[0070] The polypeptide of the present invention also includes a
polypeptide having the following properties:
[0071] (1) Action
[0072] It transfers a sulfate group from a sulfate group donor to a
hydroxyl group at 6 position of an N-acetylglucosamine residue
located at a non-reducing end of an oligosaccharide represented by
the formula I:
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I);
[0073] (2) Substrate Specificity
[0074] It does not transfer a sulfate group to chondroitin,
chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate,
keratan sulfate, desulfated keratan sulfate, CDSNS-heparin, mucin
from porcine stomach, mucin from bovine submaxillary gland, and an
oligosaccharide represented by the formula II:
Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (II);
[0075] and
[0076] (3) N-Terminal Amino Acid Sequence
[0077] It consists of an amino acid sequence represented by amino
acid numbers 1 to 48 in SEQ ID NO: 2.
[0078] For the polypeptide of the present invention,
3'-phosphoadenosine 5'-phosphosulfate (hereinafter sometimes
referred to as "PAPS") is preferably used as a sulfate group
donor.
[0079] These polypeptides of the present invention can be produced
by isolation and purification from natural source or by chemical
synthesis since their amino acid sequences and properties are
disclosed by this invention. Preferably, the polypeptide is
produced using the DNA of the present invention as will be
described later. The method of producing the polypeptide of the
present invention using the DNA of the present invention will be
described later. The polypeptide of the present invention is not
necessarily a single polypeptide but may be a part of a fusion
protein if necessary. For example, a fusion protein comprising the
polypeptide of the present invention and another polypeptide
necessary for expression may be exemplified.
[0080] Since the polypeptide of the present invention has an
activity to specifically transfer a sulfate group from a sulfate
group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end of
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc, the polypeptide of the present
invention can be used for specifically sulfating the 6 position of
an N-acetylglucosamine residue located at a non-reducing end,
synthesizing sugar chains having a GlyCAM-1 structure and so
on.
[0081] <2> The DNA of the Present Invention
[0082] The DNA of the present invention is a DNA encoding a
polypeptide of (a) or (b) below:
[0083] (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 2; or
[0084] (b) a polypeptide which comprises an amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a) and which has
an enzymatic activity to transfer a sulfate group from a sulfate
group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end of an
oligosaccharide represented by the formula I:
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I).
[0085] Among these, a DNA encoding the polypeptide (a) is
preferable.
[0086] For example of such DNA, a DNA containing a nugleotide
sequence represented by nucleotide numbers 470 to 1918 in SEQ ID
NO: 1, may be exemplified.
[0087] Furthermore this DNA has been derived from mouse originally.
As a matter of course, however, the source of the DNA of the
present invention is not limited and the DNA also includes that is
produced by genetic engineering procedure or chemical
synthesis.
[0088] The DNA of the present invention also includes a DNA
encoding a polypeptide of (a) or (b) below:
[0089] (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 4; or
[0090] (b) a polypeptide which comprises an-amino acid sequence
including substitution, deletion, insertion or transposition of one
or few amino acids in the amino acid sequence of (a) and which has
an enzymatic activity to transfer a sulfate group from a sulfate
group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end of an
oligosaccharide represented by the formula I:
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc (I)
[0091] Among these, the DNA encoding the polypeptide (a) is
preferred because the polypeptide of the present invention encoded
by the DNA has 85% or more homology to the polypeptide of the
present invention consisting of an amino acid sequence represented
by SEQ ID NO: 2.
[0092] As a specific example of such DNA, for example a DNA
comprising the nucleotide sequence represented by nucleotide
numbers 390 to 1841 in SEQ ID NO: 3, may be exemplified.
[0093] Furthermore this DNA has been derived from human originally.
As a matter of course, however, the source of the DNA of the
present invention is not limited and the DNA also includes that is
produced by genetic engineering procedure or chemical
synthesis.
[0094] Furthermore one of ordinary skilled in the art would readily
understand that the DNA of the present invention includes DNA
having the nucleotide sequence different from that as described
above due to degeneracy of the genetic codes.
[0095] The DNA of the present invention also includes DNA or RNA
complementary to the DNA of the present invention. Furthermore, the
DNA of the present invention may be either a single-stranded coding
chain encoding the polypeptide of the present invention or a
double-stranded chain consisting of the above single-stranded chain
and a DNA or a RNA having complementary nucleotide sequence
thereto.
[0096] As described in <1> above, the polypeptide of the
present invention includes polypeptides which are slightly
different in structure but are not significantly different in
function from the polypeptide of the present invention. The DNA of
the present invention also includes DNA encoding polypeptides which
are slightly different in structure but are not significantly
different in function from the polypeptide of the present
invention.
[0097] A specific example of such DNA is a DNA which is slightly
different in structure from the DNA of the present invention due to
polymorphism or mutation of DNA but which encodes a polypeptide
having the function substantially equivalent to that of the
polypeptide of the present invention.
[0098] The gene encoding the polypeptide of the present invention
derived from a chromosome is expected to contain introns in the
coding region. DNA fragments separated by introns are also included
in the DNA of the present invention as long as the fragments
encodes the polypeptide of the present invention. Namely the
meaning of the term "encode" used herein covers to have a
nucleotide sequence that undergoes processing upon transcription
and finally becomes capable of expressing a desired
polypeptide.
[0099] For the DNA of the present invention, PAPS is preferred as a
sulfate group donor.
[0100] 1. The Method of Producing the DNA of the Present
Invention
[0101] Since the nucleotide sequence of the DNA of the present
invention was revealed by the present invention, the DNA can be
synthesized based on the sequence. Alternatively, the DNA can be
obtained by amplifying the DNA of the present invention from a
chromosomal DNA or mRNA by polymerase chain reaction method (PCR)
using oligonucleotide primers prepared based on the sequence. The
DNA of the present invention was obtained for the first time by the
cDNA cloning comprising the following steps as described in the
examples below.
[0102] (1) Production of the DNA of the present invention derived
from mouse:
[0103] i) preparation of oligonucleotide primers for PCR;
[0104] ii) amplification by reverse transcription PCR (RT-PCR)
using a mouse total RNA;
[0105] iii) screening the DNA of the present invention from a mouse
cDNA library using the PCR products.
[0106] (2) Production of the DNA of the present invention derived
from human:
[0107] i) screening the DNA of the present invention from a human
cDNA library using the cDNA obtained in iii) of (1) above;
[0108] ii) Sequencing nucleotide sequence of the cDNA thus
obtained.
[0109] But the method of producing the DNA of the present invention
is not to be limited to the above method. The DNA of the present
invention can be prepared by other known cDNA cloning methods.
[0110] An example of the method of producing the DNA of the present
invention is described in detail below.
[0111] (1) Preparation of Oligonucleotide Primers for PCR
[0112] Oligonucleotide primers (a sense primer and an antisense
primer) are prepared based on a mouse expressed sequence tag (EST)
sequence (Genbank accession number, AA103962) having homology to
the catalytic site of mouse chondroitin 6-sulfotransferase.
Specific examples as oligonucleotide primers are
[0113] 5'-GTCGTCGGACTGGTGGACGA-3' (SEQ ID NO: 5) as a sense primer
and
[0114] 5'-CCCAGAGCGTGGTAGTCTGC-3' (SEQ ID NO: 6) as an antisense
primer respectively. These are also preferably used.
[0115] (2) Amplification by RT-PCR using a Mouse Total RNA as a
Template
[0116] A total RNA can be obtained by a known method (e.g.,
Kingston, R. S., (1991) in Current Protocols in Molecular Biology,
Suppl. 14, Unit 4.2, Greene Publishing Associates and Wiley
Interscience, New York). Any starting material can be used without
limitation as long as it expresses mRNA of the polypeptide of the
present invention. For example, mouse embryo, particularly
13-day-old embryo can be used.
[0117] A partial cDNA encoding the polypeptide of the present
invention can be amplified by RT-PCR using the above total RNA as a
template and oligonucleotide primers. PCR can be performed
following the usual method.
[0118] (3) Screening the DNA of the Present Invention from a Mouse
cDNA Library using the PCR Products
[0119] The PCR products obtained in RT-PCR of (2) above are
labelled with .sup.32P or the like, then used as hybridization
probes for screening cDNA fragments (the DNA of the present
invention) from a cDNA library. The mouse cDNA library to be used
is not particularly limited. As an example thereof, a
.lambda.gt11-library containing mouse embryo cDNA (.lambda.gt11
mouse embryo cDNA library) may be exemplified.
[0120] Hybridization can be carried out by a known method (e.g., J.
Biol. Chem., 270, 18575-18580 (1995)).
[0121] A DNA insert is isolated from a positive clone by digestion
with EcoRI and subcloned into, for example, pBluescript II SK-
(STRATAGENE). Thereafter, the nucleotide sequence can be determined
by a known method such as the dideoxy chain termination method
(Proc. Natl. Acad. Sci. U.S.A., 74, 5463-5467 (1977)).
[0122] The nucleotide sequence of the DNA of the present invention
obtained by a series of the above methods and the amino acid
sequence deduced from this nucleotide sequence are shown in SEQ ID
NO: 1 and the same amino acid sequence alone is shown in SEQ ID NO:
2.
[0123] (4) Screening a Human cDNA Library and Sequencing of
Nucleotide Sequence using the (Mouse) cDNA as Obtained Above
[0124] The human DNA of the present invention can be obtained by
screening a human cDNA library (e.g., a .lambda.gt11 library
containing human fetal brain cDNA) using the (mouse) cDNA as
obtained above. The nucleotide sequence can also be determined by
the method as described above.
[0125] The nucleotide sequence of the DNA of the present invention
obtained by this method and the amino acid sequence deduced from
this nucleotide sequence are shown in SEQ ID NO: 3 and the same
amino acid sequence alone is shown in SEQ ID NO: 4.
[0126] 2. The Method of Producing the Polypeptide of the Present
Invention Using the DNA of the Present Invention
[0127] Cells into which the DNA of the present invention has been
transfected are cultivated in a suitable medium to allow the
polypeptide encoded by the DNA of the present invention to be
produced and accumulated in the culture. The polypeptide of the
present invention can be produced by recovering it from the
culture.
[0128] The cells into which the DNA of the present invention has
been transfected can be obtained by inserting the fragment of the
DNA of the present invention into a known expression vector to
construct a recombinant plasmid and transfecting the recombinant
plasmid into the cells. The DNA of the present invention used
herein is not particularly limited as long as it is the DNA of the
present invention. It is preferable to use a DNA comprising the
nucleotide sequence represented by nucleotide numbers 470 to 1918
in SEQ ID NO: 1 and particularly preferable to use a DNA having the
nucleotide sequence represented by nucleotide numbers 467 to 1921
in SEQ ID NO: 1. It is also preferable to use a DNA comprising the
nucleotide sequence represented by nucleotide numbers 390 to 1841
in SEQ ID NO: 3 and particularly preferable to use a DNA comprising
the nucleotide sequence represented by nucleotide numbers 387 to
1844 in SEQ ID NO: 3.
[0129] As the cells, procaryotic cells such as Escherichia coli and
eucaryotic cells such as mammalian cells may be exemplified. When
procaryotic cells such as Escherichia coli are used, addition of
sugar chain does not occur to the polypeptide produced by
expression of the DNA of the present invention, then the
polypeptide of the present invention having no sugar chain can be
obtained. When eucaryotic cells such as mammalian cells are used,
sugar chain may add to the polypeptide produced by expression of
the DNA of the present invention, then the form of the polypeptide
of the present invention comprising sugar chain can be
obtained.
[0130] In this producing method, a host-vector system usually used
for production of proteins can be used. For example, it is
preferable to use a combination of a cultured cell derived from
mammals such as COS-7 cells and an expression vector for mammalian
cells such as pcDNA3 expression vector (Invitrogen Co.). Culture
media and culturing conditions are appropriately selected depending
on the host, that is cells, to be used.
[0131] The DNA of the present invention may be expressed directly.
Alternatively, it may be expressed with another polypeptide as a
fusion polypeptide. The full-length DNA of the present invention
may be expressed. It may also be expressed in part as a partial
peptide.
[0132] Recovering the polypeptide of the present invention from the
culture product may be performed by known extraction and
purification methods for polypeptides. The culture product used
herein includes the medium and the cells in the medium.
[0133] Specific examples of the method of extracting the
polypeptide of the present invention include extraction by
disrupting cells such as homogenization, glass beads mill method,
sonication, osmotic shock procedure, and freezing and thawing
method, extraction with a surfactant, or any combination
thereof.
[0134] For example, homogenization is preferable when the
polypeptide of the present invention is extracted from the
above-described culture product.
[0135] More specifically, the cells are collected from the culture
product, and a buffer (which may contain a surfactant) is added
thereto. The resulting cell suspension is homogenized with a
homogenizer and then extraction can be performed by separating it
into cell residue and a supernatant (extract) by a separation
method such as centrifugation and it is prefarable.
[0136] Specific examples of the method of purifying the polypeptide
of the present invention include salting out with salt such as
ammonium sulfate or sodium sulfate, centrifugation, dialysis,
ultrafiltration, absorption chromatography, ion exchange
chromatography, hydrophobic chromatography, reverse phase
chromatography, gel filtration, gel permeation chromatography,
affinity chromatography, electrophoresis, and any combination
thereof.
[0137] It can be confirmed whether the polypeptide of the present
invention has been produced or not by analyzing amino acid
sequence, action, and substrate specificity of the purified
polypeptide and comparing with physical properties of the
polypeptide of the present invention as described in <1>.
[0138] The DNA of the present invention would be expected to used
for production of the polypeptide of the present invention in large
scale, artificial synthesis of GlyCAM-1 in the living organisms
(cells), or the like.
[0139] <3> The Producing Method 1 of the Present
Invention
[0140] The producing method 1 of the present invention is a method
of producing a sulfated sugar represented by the formula III, which
comprises a step of reacting the polypeptide of the present
invention with a sugar chain represented by the formula IV.
(SO.sub.4-6)GlcNAc-R (III),
GlcNAc-R (IV),
[0141] wherein --R represents a hydrogen atom or a sugar residue
bonded by a glycosidic linkage.
[0142] The polypeptide of the present invention usable in the
producing method 1 of the present invention is not particularly
limited as long as it is the polypeptide of the present invention.
The polypeptide of the present invention usable herein is not
necessarily completely purified as long as the activities of the
polypeptide are not substantially deteriorated. Thus, the
polypeptide partially purified or in the form of the cell extract
may also be used.
[0143] By the producing method 1 of the present invention, the
polypeptide of the present invention has been found to have an
enzymatic activity to specifically transfer a sulfate group from a
sulfate group donor to a hydroxyl group at 6 position of an
N-acetylglucosamine residue located at a non-reducing end. The
producing method 1 of the present invention is a method of
producing a sulfated sugar utilizing the above finding. The most
important and essential element in the production method 1 of the
present invention is the part except --R in the above formula III
and IV. In other words, --R may have an optional structure and the
structure of the sugar residue is not particularly limited. For
example, --R may be a sugar-chain (glycolipid) having a lipid (such
as a ceramide residue) at its end.
[0144] The above formulae III and IV are preferably the sugar
chains represented by the formulae V and VI, respectively.
(SO.sub.4-6)GlcNAc.beta.1-3Gal.beta.1-4GlcNAc-R' (V)
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc-R' (VI),
[0145] wherein --R' represents a hydrogen atom or a sugar residue
bonded a glycosidic linkage.
[0146] R' is preferably a hydrogen atom or a sugar chain having 1
to 15 saccharides, more preferably a hydrogen atom or a sugar chain
having 1 to 10 saccharides, particularly preferably a hydrogen atom
or a sugar chain having 1 to 2 saccharides, and most preferably a
hydrogen atom. When --R' is a sugar chain, it has preferably a
backbone composed of a repeating structure of
(-3Gal.beta.1-4GlcNAc.beta.1-) (lactosamine). In this case, a
sialic acid residue, a fucose residue, a sulfate group and so on
may be added to the backbone of --R'.
[0147] When the polypeptide of the present invention is reacted
with the sugar chain represented by the formula IV in the producing
method 1 of the present invention, it is preferable to coexist with
a sulfate group donor.
[0148] In the producing method 1 of the present invention, PAPS is
preferably used as a sulfate group donor.
[0149] The reaction to subject the polypeptide of the present
invention to the sugar chain represented by the formula IV can be
carried out by making coexist with the polypeptide of the present
invention, the sugar chain represented by the formula IV, and the
sulfate group donor. The pH in this reaction is not particularly
limited as long as the activity of the polypeptide of the present
invention can be retained. The reaction is preferably performed at
around the neutral pH (for example, about pH 6.8), more preferably
in a buffer solution having the buffering activity at this pH
value. The reaction temperature is not particularly limited as long
as the activity of the polypeptide of the present invention can be
retained. The temperature from about 30 to 40.degree. C. may be
exemplified. Furthermore, when some substance enhances the activity
of the polypeptide of the present invention, the substance may be
added. One of ordinary skilled in the art would determine the
reaction time depending on amounts used of the sugar chain, the
sulfate group donor, and the polypeptide of the present invention
and other reaction conditions thereof. In the reaction, MnCl.sub.2
or the like may coexist.
[0150] In the case of small scale production, the polypeptide of
the present invention can exist under the coexistence with the
sugar chain represented by the formula IV and the sulfate group
donor and said polypeptide may be reacted. In the case of large
scale production, the polypeptide of the present invention can be
continuously reacted by using immobilized enzyme prepared by
immobilizing the polypeptide of the present invention with an
appropriate solid phase (beads, or the like) or a membrane reactor
such as a ultrafiltration membrane, or a dialysis membrane. A
bioreactor for regenerating (synthesizing) the sulfate group donor
may be used in combination.
[0151] By the action of the polypeptide of the present invention, a
sulfate group is specifically transfected to a hydroxyl group at 6
position of the N-acetylglucosamine residue located at the
non-reducing end of the sugar chain represented by the formula IV
to thereby form the sugar chain represented by the formula III.
[0152] The sugar chain represented by the formula III can be
recovered from the reaction mixture by usual methods for isolating
and purifying sugar chains. Examples of such methods include
adsorption chromatography, anion exchange chromatography,
hydrophobic chromatography, gel filtration, gel permeation
chromatography, paper electrophoresis, paper chromatography, thin
layer chromatography, fractionation with organic solvents
(preferably alcohol, acetone, and the like), and any combination of
these methods. However, the recovering methods are not limited
thereto.
[0153] The thus-obtained sugar chain represented by the formula III
can be used as an intermediate for production of, for example,
GlyCAM-1 or its sugar chain backbone structure.
[0154] <4> The Producing Method 2 of the Present
Invention
[0155] The producing method 2 of the present invention is a method
of producing sulfated sugar which comprises steps of transfecting
cells with the DNA of the present invention and then cultivating
the cells.
[0156] As long as the method includes at least these steps, any
method comprising additional steps is included in the producing
method 2 of the present invention. For example, the producing
method 2 of the present invention includes a method of producing
sulfated sugar which comprises cultivating cells transfected with
the DNA of the present invention in an appropriate culture medium,
isolating the cells from the culture product, and collecting
sulfated sugar chain expressed on the cells.
[0157] The produced sulfated sugar is not necessarily isolated and
purified from the cells. When the cells themselves on whose surface
sulfated sugar is expressed are desired, the cells are collected in
the culture product and used as they are.
[0158] The DNA of the present invention can be transfected into
cells by a known method using DEAE-dextran, calcium phosphate,
polybrene, and the like or the other method well known in genetic
engineering field.
[0159] The DNA of the present invention to be transfected into
cells is preferably transfected into the cells in the form of a
recombinant plasmid prepared by inserting the DNA of the present
invention into a known expression vector. The DNA of the present
invention used herein is not particularly limited as long as it is
included in the DNA of the present invention. It is preferable to
use a DNA comprising the nucleotide sequence represented by
nucleotide numbers 470 to 1918 in SEQ ID NO: 1, particularly a DNA
having the nucleotide sequence represented by nucleotide numbers
467 to 1921 in SEQ ID NO: 1.
[0160] Procaryotic cells such as Escherichia coli and eucaryotic
cells such as mammalian cells are exemplified as the cells.
[0161] In this producing method, a host-vector system usually used
for production of proteins can be used. For example, it is
preferable to use a combination of a cultured cell derived from
mammals such as COS-7 cells and an expression vector for mammalian
cells such as pcDNA3 expression vector (Invitrogen Co.). Culture
media and culturing conditions are appropriately selected depending
on the host, that is cells, to be used. The culture product can be
obtained by performing cultivation under such conditions.
[0162] The sulfated sugar may be collected from the culture product
by known extraction and purification methods for glycolipids. The
culture product used herein includes the medium and the cells in
the medium.
[0163] Specific examples of the method of extracting glycolipids
includes extraction with an organic solvent such as methanol or
chloroform, extraction by disrupting cells such as homogenization
or sonication, and any combination thereof. The cells in the
culture product are subjected to such extraction procedures to
obtain extract containing sulfated sugar.
[0164] The sulfated sugar can be isolated and purified from the
extract by usual methods for isolating and purifying sugar chains.
It can be performed by the method such as adsorption
chromatography, anion exchange chromatography, hydrophobic
chromatography, gel filtration, gel permeation chromatography,
paper electrophoresis, paper chromatography, thin layer
chromatography, fractionation with organic solvents (preferably
alcohol, acetone, and the like), and any combination of these
methods. However, the recovering methods are not limited
thereto.
[0165] Through the step that the hydroxyl group at 6th position of
the N-acetylglucosamine residue located at the non-reducing end of
the sugar chain is sulfated by the action of the polypeptide of the
present invention expressed by transfecting the DNA of the present
invention into the cells, the sulfated sugar produced by the
producing method 2 of the present invention is expressed on the
cell surface. The sulfated sugar expressed has at least a sugar
chain structure represented by the formula VII:
NeuAc.alpha.2-3Gal.beta.1-4(So.sub.4-6)GlcNAc-R,
[0166] wherein --R represents a hydrogen atom or a sugar residue
bonding by a glycosidic linkage. A lipid such as ceramide may be
bound to the hydrogen atom or the sugar residue bonding by a
glycosidic linkage.
[0167] This sugar chain structure is recognized by antibodies that
reacts with 6-sulfated sialyl Lewis X ceramide (6-Sulfo SLeX
ceramide) and SL2L4
(NeuAc.alpha.2-3Gal.beta.1-4(SO.sub.4-6)GlcNAc.beta.1-3(SO.sub.4-6)Gal.be-
ta.1-4(SO.sub.4-6)GlcNAc) but does not react with 6'-sulfated
sialyl Lewis X ceramide (6'-Sulfo SLeX ceramide) and
sialylparagloboside (SPG) (the minimum structure necessary for the
reaction is NeuAc.alpha.2-3Gal.beta.1- -4(SO.sub.4-6)GlcNAc). Such
antibodies can be prepared by usual methods for preparing
antibodies using 6-sulfated sialyl Lewis X ceramide. Antibodies are
preferably monoclonal antibodies.
[0168] The monoclonal antibodies can be prepared by the method of
Kohler and Milstein (Nature 256, 495-497 (1975)). For example,
6-sulfated sialyl Lewis X ceramide is allowed to be adsorbed to
bacterial cells such as Salmonella minnesota. The resulting
conjugate is administered to an animal to be immunized such as
mice, rats, guinea pigs, rabbits, goats, sheep, and the like,
intraperitoneally, subcutaneously, or into footpads. Spleen or
popliteal lymph nodes are taken out and cells collected from these
tissues are subjected to cell fusion with a tumor cell line,
myeloma, to establish hybridoma. The hybridomas thus obtained are
subcultured to be selected among these hybridoma continuously
producing an antibody that reacts with 6-sulfated sialyl Lewis X
ceramide and SL2L4 but does not react with 6'-sulfated sialyl Lewis
X ceramide and sialylparagloboside (an antibody having as the
minimum structure necessary for the reaction of
NeuAc.alpha.2-3Gal.beta.1-4(So.sub.4-6)GlcN- Ac). The thus-selected
cell line is cultured in an appropriate medium and a monoclonal
antibody is produced in the medium. Alternatively, the monoclonal
antibody can be produced in large scale by culturing the
above-described hybridoma in vivo, for example, in abdominal cavity
of a mouse. As the cells to be used for cell fusion, lymph node
cells and lymphocytes in peripheral blood as well as spleen cells
can be used. Myeloma cell line is preferably derived from the same
cell line as compared with-those derived from heterologous cell
line so as to obtain a hybridoma that stably produces antibodies.
An example of the antibody includes the G72 antibody as will be
described in Examples later.
[0169] The sulfated sugar produced by the producing method 2 of the
present invention can be confirmed for its production by using the
above-described antibody. A known immunological method can be used
for confirming the production using the antibody. For example, such
a method includes immunoblotting, labeling immunoassay (e.g., EIA,
ELISA, radioimmunoassay, fluoroimmunoassay, or the like). When the
sulfated sugar expressed on the cell surface is to be confirmed,
flow cytometry can also be used. As a matter of course, the
sulfated sugar produced by the producing method 2 of the present
invention can be confirmed by using a known technique for analyzing
sugar chain structures.
[0170] In the producing method 2 of the present invention, cDNA
encoding fucosyltransferase can be transfected into cells together
when the DNA of the present invention is transfected into cells.
Thus, the producing method 2 of the present invention includes a
method of producing sulfated sugar which comprises steps of
transfecting cells with the DNA of the present invention and cDNA
encoding fucosyltransferase concurrently and then cultivating the
cells.
[0171] Fucosyltransferase to be transfected is not particularly
limited. It is known that fucosyltransferase IV can form the Lewis
X structure (Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc) by transferring
fucose-to N-acetyllactosamine (J. Biol. Chem. 266, 17467-17477
(1991), Cell 63, 1349-1356 (1990)) and thus is preferably used. The
cDNA encoding fucosyltransferase can be inserted into an
appropriate expression vector and transfected into cells
concurrently with the DNA of the present invention. Preferable
expression vectors are those used for the DNA of the present
invention.
[0172] The sulfated sugar produced by this method is expressed on
the cell surface through the process in which the hydroxyl group at
6 position of the N-acetylglucosamine residue located at the
non-reducing end of the sugar chain is sulfated by the actions of
the polypeptide of the present invention expressed by the DNA of
the present invention inserting into the cells, fucosyltransferase
and then fucose is transferred to N-acetyllactosamine by the
actions of fucosyltransferase expressed by the cDNA of
fucosyltransferase inserting into the cells. The expressed sulfated
sugar has at least a sugar chain structure represented by the
formula VIII;
Gal.beta.1-4(Fuc.alpha.1-3)(SO.sub.4-6)GlcNAc-R (VIII)
[0173] wherein --R represents a hydrogen atom or a sugar residue
bonding by a glycosidic linkage. A lipid such as ceramide may be
bound to the hydrogen atom or the sugar residue bonding by a
glycosidic linkage.
[0174] This sugar chain structure is recognized by monoclonal
antibodies that specifically react with 6-sulfated Lewis X
(Gal.beta.1-4(Fuc.alpha.1- -3) (SO.sub.4-6)GlcNAc; 6-Sulfo LeX)
structure but do not react with Lewis X
(Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc; LeX) and 6'-sulfated Lewis X
((SO.sub.4-6)Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc; 6'-sulfo LeX)
structures. Such antibodies can be prepared by usual methods for
preparing antibodies using 6-sulfated Lewis X ceramide
(Gal.beta.1-4 (Fuc.alpha.1-3) (SO.sub.4-6)
GlcNAc.beta.1-3Gal.beta.1-4Glc.beta.1-Cer) Antibodies are
preferably monoclonal antibodies.
[0175] The monoclonal antibodies can be prepared by the method as
described above. An antigen to be used for immunization is
preferably 6-sulfated Lewis X ceramide adsorbed to bacterial cells
such as Salmonella minnesota.
[0176] From the hybridomas prepared by the above method, cell lines
continuously producing an antibody that specifically reacts with
6-sulfated Lewis X structure but does not react with Lewis X and
6'-sulfated Lewis X are selected. A monoclonal antibody is obtained
in the medium by culturing the thus-selected cell line in an
appropriate medium. Alternatively, the monoclonal antibody can be
produced in large scale by culturing the above-described hybridoma
in vivo, for example, in abdominal cavity of a mouse. The cells to
be used for cell fusion are also the same as described above.
[0177] An example of such an antibody includes the AG223 antibody
as will be described in Examples later.
[0178] The sulfated sugar produced by this method can be confirmed
for its production by using the above-described antibody.
Confirmation with antibodies can be performed by using known
immunological techniques. As a matter of course, it can be
confirmed using a known technique for analyzing sugar chain
structure. These methods can be explained as described above.
[0179] The obtained sugar chains represented by the formulae VII
and VIII can be used as intermediates for production of, for
example, GlyCAM-1 or its sugar chain backbone structure.
[0180] The polypeptide of the present invention is a polypeptide of
N-acetylglucosamine-6-O-sulfotransferase having activity to
specifically transfer a sulfate group to a hydroxyl group at 6
position of an N-acetylglucosamine residue located at a
non-reducing end of a sugar chain. Therefore, it is useful for
synthesis of functional sugar chains such as GlyCAM-1 (expected to
be used as an anti-inflammatory agent or the like). The DNA of the
present invention can be used for synthesis of the polypeptide of
the present invention in large scale and artificial expression of
functional sugar chains such as GlyCAM-1 in the living organism
(cells).
[0181] The methods 1 and 2 of the present invention is useful as a
method of producing functional sugar chains such as GlyCAM-1 or
their synthetic intermediates.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0182] Hereinafter, the present invention will be more specifically
explained with reference to Examples.
[0183] <1> Materials and Methods Commonly used Throughout the
Examples
[0184] (1) Materials
[0185] .sup.35S-PAPS (3'-Phosphoadenosine-5'-phosphosulfate; 58.1
GBq/mmol) was from DuPont NEN; .sup.3H-NaBH.sub.4(16.3 GBq/mmol)
and .alpha.-.sup.32P-dCTP (110 GBq/nmol) were from Amersham;
chodroitin sulfate A (whale cartilage), chondroitin sulfate C
(shark cartilage), dermatan sulfate, completely desulfated and
N-resulfated heparin (CDSNS-heparin) and Streptococcus
.beta.-galactosidase were from Seikagaku Corporation; unlabeled
PAPS, mucin from porcin stomach and mucin from bovine submaxillary
gland were from Sigma; Hiload Superdex 30 HR 16/60 and fast
desalting column HR 10/10 were from Pharmacia Biotech; Partisil
SAX-10 was from Whatman; the mouse day-7 embryo 5'-STRETCH PLUS
.lambda.gt 11 cDNA library was from CLONTECH; an anti-Lewis X
antibody LeuM1 was from Becton Dickinson Labware.
[0186] Keratan sulfate from bovine cornea and
Gal.beta.1-4GlcNAc.beta.1-3G- al.beta.1-4GlcNAc (sometimes referred
to as "L1L1" herein) were from Seikagaku Corporation. Partially
desulfated keratan sulfate (sulfate/glucosamine=0.62) was prepared
from corneal keratan sulfate as described (J. Biol. Chem., 272,
32321-32328 (1997), J. Biochem. (Tokyo) 86, 1323-1329 (1979)).
[0187] GlcNAc.beta.1-3Gal.beta.1-4GlcNAc was prepared from L1L1 by
.beta.-galactosidase digestion. Reaction mixture for
.beta.-galactosidase digestion contained 2 mg L1L.sub.1, 5 .mu.mole
of sodium acetate buffer, pH 5.5, and 40 mU enzyme in a final
volume of 100 .mu.l (J. Biochem. (Tokyo) 80, 9-17 (1976)). The
reaction mixture was incubated at 37.degree. C. for 24 h. After
digestion with .beta.-galactosidase,
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc was purified by Superdex 30
chromarograhy and desalted by lyophilization.
Gal.beta.1-4-3H--(SO.sub.4-- 6)2,5-anhydromannitol and
(SO.sub.4-6)2, 5.sup.3H-anhydromannitol were obtained from keratan
sulfate by reaction sequence of N-deacetylation, deamination,
NaB.sup.3H.sub.4 reduction and partial acid hydrolysis
(Glycobiology, 6, 51-57 (1996), Biochem. J. 235, 225-236
(1986)).
[0188] Structere of synthetic glycolipids used in the examples as
follows; sialyl Lewis X
ceramide=NeuAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc.-
beta.1-3Gal.beta.1-4Glc.beta.1-Cer; 6-sulfo sialyl Lewis X
ceramide=NeuAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3) (SO.sub.4-6)
GlcNAc.beta.1-3Gal.beta.1-4Glc.beta.1-Cer; 6'-sulfo sialyl Lewis X
ceramide=NeuAc.alpha.2-3(SO.sub.4-6)Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc
.beta.1-3Gal.beta.1-4Glc.beta.1-Cer; 6,6'-bis-sulfo sialyl Lewis X
ceramide=NeuAc.alpha.2-3(SO.sub.4-6)Gal.beta.1-4(Fuc.alpha.1-3)(SO.sub.4--
6)GlcNAc.beta.1-3Gal.beta.1-4Glc.beta.1-Cer (Carbohydr. Res., 209,
c1-c4 (1991), Carbohydr. Res., 285, c1-c8 (1996), J. Med. Chem.,
39, 1339-1343 (1996)). These glycolipids were generous gifts from
Dr. Makoto Kiso, Faculty of Agriculture, Gifu University. The
asialo compounds were prepared from the corresponding synthetic
glycolipids by digestion with neuramimidase from Arthrobacter
ureafaciens (Nakarai Tesque Co.). The sialylated paraglobosides
(NeuAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.-
beta.1-4Glc.beta.1-Cer and
NeuAc.alpha.2-6Gal.beta.1-4GlcNAc.beta.1-3Gal.b-
eta.1-4Glc.beta.1-Cer) were prepared from human colon and liver
carcinoma tissues.
NeuAc.alpha.2-3Gal.beta.1-4(SO.sub.4-6)GlcNAc.beta.1-3(SO.sub.4--
6)Gal.beta.1-4(SO.sub.4-6)GlcNAc (sometimes referred to as "SL2L4"
herein) and
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4GlcNAc were
generous gifts from Dr. Keiichi Yoshida (Seikagaku Corporation) The
former was coupled to cholesteryl aniline through reductive
amination (Blood, 82, 2797-2805 (1993)).
[0189] (2) Isolation of the DNA of the Present Invention
[0190] (2-1) Isolation of the DNA of the Present Invention Derived
from Mouse
[0191] A mouse expressed sequence tag (EST) sequence (Genbank
accession number AA103962) with similarity to the catalytic portion
of mouse chondroltin 6-sulfotransferase was amplified by the RT-PCR
method using mouse day-13 embryo total RNA as a template. The sense
primer, GTCGTCGGACTGGTGGACGA (SEQ ID NO:5) and the antisense
primer, CCCAGAGCGTGGTAGTCTGC (SEQ ID NO:6), were used for PCR
amplification, which was carried out at 94.degree. C. for 3 min,
with 35 cycles of 94.degree. C. for 0.5 min, 60.degree. C. for 1
min and 72.degree. C. for 1 min. The PCR product (368 bp) was
.sup.32P-labeled with a Megaprime.TM. DNA labeled kit (Amersham
Co.) and was used to screen the .lambda.gt 11 mouse day-7 embryo
cDNA library.
[0192] Hybridization was carried out as described (J. Biol. Chem.,
270, 18575-18580 (1995)). DNA insert was isolated from positive
.lambda.gt11 clones by digestion with EcoRI and was subcloned into
the pBluescriptII SK-(STRATAGENE Co.). Then, the nucleotide
sequence was determined by the dideoxy chain termination method
(Proc. Natl. Acad. Sci. U.S.A., 74, 5463-5467 (1977)) using an
Applied Biosystems automated sequencer.
[0193] (2-2) Isolation of the Human DNA of the Present
Invention
[0194] The DNA of the present invention derived from mouse as
obtained above was labeled with .sup.32P using Megaprime DNA
labeling systems (Amersham Co.). Using this as a probe, the
.lambda.gt11 library containing cDNA of human fetal brain (CLONTECH
CO.) was screened. Hybridization was carried out by the method
described in J. Biol. Chem. 272, 32321-32328 (1997).
[0195] A DNA insert was isolated from positive .lambda.gt11 clones
by digestion with EcoRI and subcloned into pBluescript II SK-
(STRATAGENE CO.). The nucleotide sequence was determined by the
method used for the above-described DNA of the present invention
derived from mouse.
[0196] (3) Construction of Expression Vectors
[0197] A cDNA fragment encoding the open reading frame of the
polypeptide of the present invention (the DNA of the present
invention derived from mouse) was amplified by PCR using the cloned
cDNA fragment from mouse as a template. The sense primer,
ACGAATTCGGGATGAAGGTATTTCGCAGG (SEQ ID NO:7), and the antisense
primer, ATGAATTCTCAAAGCCGGGGCTTCCTGAG (SEQ ID NO:8), were used for
PCR amplification, which was carried out at 94.degree. C. for 3
min, with 35 cycles of 94.degree. C. for 1 min, 60.degree. C. for 1
min and 72.degree. C. for 2 min in 5% (v/v) dimethylsulfoxide. The
PCR product including the open reading frame of polypeptide of the
present invention (nucleotide numbers 467 to 1921 in SEQ ID NO:1)
was digested with EcoRI and subcloned into the pcDNA3 expression
vector (Invitrogen Co.). Recombinant plasmids containing the DNA
fragment in the correct orientation, pcDNA3-GlcNAc6ST, was used for
expression. The recombinant plasmids containing the DNA fragment in
the reverse orientation, pcDNA3-GlcNAc6STA, was used in control
expetiments.
[0198] An expression vector was constructed in the same manner as
described above using the DNA of the present invention derived from
human. First, PCR was carried out using CTGAATTCGGAATGAAGGTGTTCCGTA
(SEQ ID NO: 9) and GAGAATTCTTAGAGACGGGGCTTCCGA (SEQ ID NO: 10) as
primers, and the cloned human cDNA fragment as a template to obtain
PCR products (nucleotide numbers 387 to 1844 in SEQ ID NO: 3)
containing an open reading frame of the polypeptide of the present
invention. The PCR products was digested with EcoRI and subcloned
into the expression vector, pcDNA3. A recombinant plasmid
(pcDNA3-hGlcNAc6ST) containing the DNA fragment in the correct
orientation was used for expression, while a recombinant plasmid
(pcDNA3-hGlcNAc6STA) containing the DNA fragment in the reverse
orientation was used as control.
[0199] Previously cloned 1544 bp fragment of the mouse
fucosyltransferase IV gene (J. Biochem. (Tokyo) 119, 302-308
(1996)) was subcloned into the BamHI and EcoRI sites of the pcDNA3
expression vector as described above. The recombinant plasmid
containing the DNA fragment in the correct orientation,
pcDNA3-FucTIV, was used.
[0200] (4) Transient Expression of the DNA of the Present Invention
in COS-7 Cells
[0201] COS-7 cells (3.times.10.sup.6 cells in a 10 cm-dish, from
Riken cell bank) were transfected with 15 .mu.g of expression
plasmids by the DEAE-dextran method (Aruffo, A. (1991) in Current
Protocols in Molecular Biology, Suppl. 14, Unit 16.13, Greene
Publishing Associates and Wiley Interscience, New York). After 65 h
culture in Dulbecco-modified minimum essential medium containig 10%
fetal calf serum, the cells were washed with phosphate buffered
saline (PBS) and scraped off the dishes. These cells were collected
and homogenized with a Dounce homogenizer in 1.5 mL/dish of 0.25 M
sucrose, 10 mM Tris-HCl, pH 7.2, and 0.5% Triton X-100. The
homogenates were centrifuged at 10,000.times. g for 15 min, and the
supernatant was saved. This supernatant is hereinafter referred to
as "extracts". For FACS (fluorescence-activated cell sorter)
analysis, the transfected cells were cultured for 48 h, transferred
into 25 cm.sup.2 culture flasks (3.times.10.sup.5 cells per flask)
and further cultured for 36 h.
[0202] (5) Assay of Sulfotransferase Activity to Various High
Molecular Weight Substrates
[0203] Sulfotransferase activities were assayed using various
glycosaminoglycans as substrates (acceptors) as described (J. Biol.
Chem., 272, 32321-32328 (1997)). When mucins were used as
acceptors, reaction mixture containing 2.5 .mu.mol of
imidazole-HCl, pH 6.8, 0.25 .mu.mol of CaCl.sub.2, 0.1 .mu.mol of
dithiothreitol, 0.1 mmol of NaF, 0.1 .mu.mol of AMP, 2.0 .mu.g of
mucins, 50 pmol of .sup.35S-PAPS (about 5.0.times.10.sup.5 cpm),
and 5 .mu.l of the extracts in a final volume of 50 .mu.l was
incubated at 37.degree. C. for 1 h.
[0204] After isolation with a fast desalting column, radioactivity
incorporated into mucins was determined.
[0205] (6) Assay of Sulfotransferase Activity to
Oligosaccharides
[0206] The reaction mixture contained 2.5 .mu.mol of imidazole-HCl,
pH 6.8, 0.5 .mu.mol of MnCl.sub.2, 0.1 .mu.mol of AMP, 1.0 .mu.mol
of NaF, 25 mmol of oligosaccharides, 50 pmol of .sup.35S-PAPS
(about 5.times.10 cpm), and 5 .mu.l of the extracts in a final
volume of 50 .mu.l. The reaction mixture was incubated at
30.degree. C. for 5 h and the reaction was stopped by immersing the
reaction tubes in a boiling water bath for 1 min. .sup.35S-Labeled
oligosaccharides were separated from .sup.35SO.sub.4 and
.sup.35S-PAPS by Superdex 30 gel chromatography, and the
radioactivity was determined. When
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc was used as an acceptor,
sulfotransferase reaction proceeded linearly up to 5 h under the
assay conditions.
[0207] (7) Superdex 30 Chromatography, Paper Electrophoresis, Paper
Chromatography, HPLC and TLC
[0208] Hiload Superdex 30 16/60 column was equilibrated with 0.2M
NH.sub.4HCO.sub.3. The flow rate was 1 ml/min. One ml fractions
were collected and mixed with 4 ml Cleasol (Nakarai Tesque Co.),
and the radioactivity was determined. Oligosaccharides were
monitored by absorption at 210 nm. Paper electrophoresis was
carried out on Whatman No. 3 paper (2.5 cm.times.57 cm) in
pyridine/acetic acid/water (1:10:400, by volume, pH 4) at 30 V/cm
for 40 min. Samples for paper chromatography was spotted on a
Whatman No. 3 paper (2.5 cm.times.57 cm) and developed with
1-butanol/acetic acid/1M NH.sub.4OH (3:2:1, by volume). The dried
paper strips after paper electrophoresis or paper chromarography
were cut into 1.25 cm segments and radioactivity was determined by
liquid scintillation counting. HPLC analysis of .sup.35S-labeled
product obtained from .sup.35S-labeled sulfated
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc was carried out on a Partisil
SAX-10 column (4.5 mm.times.25 cm) equilibrated with 5 mM
KH.sub.2PO.sub.4. The column was developed with 5 mM
KH.sub.2PO.sub.4. The flow rate was 1 ml/min and the column
temperature was 40.degree. C. Fractions, 0.5 ml, were collected and
mixed with 4 ml Clearsol, and the radioactivity was determined. TLC
was performed on aluminium sheets precoated with cellulose 0.1 mm
thick (Merck Co.) in ethyl
acetate/pyridine/tetrahydrofuran/water/acetic acid (50:22:15:15:4,
by volume) (Biochem. J., 319, 209-216 (1996)).
[0209] (8) N-Deacetylation, Deamination and NaBH.sub.4 Reduction of
the .sup.35S-labeled Sulfated GlcNAc.beta.1-3Gal.beta.1-4GlcNAc
[0210] The .sup.35S-labeled sulfated
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc was prepared using the expressed
polypeptide of the present invention (2.1 .mu.g as protein in the
extract) as described above except that concentration of
.sup.35S-PAPS was increased to 6-fold and incubation was carried
out for 25 h. The .sup.35S-labeled sulfated
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc eluted from the Superdex 30
column was lyophilized, purified by paper electrophoresis and
deacetylated with 70% hydrazine containing 1.0% hydrazine sulfate
at 95.degree. C. for 6 h (Anal. Biochem. 176, 96-104 (1989)). The
deacetylated materials were purified by Superdex 30 chromatography,
subjected to deamination with nitrous acid at pH 4 and reduced by
NaBH.sub.4(Biochem. J. 235, 225-236 (1986)). Finally the sample was
dissolved in 60 .mu.l of water and subjected to paper
chromatography.
[0211] (9) Immunological Methods
[0212] A hybridoma cell line, AG223, which secreted murine IgM
monoclonal antibody (AG223) reactive with 6-sulfo Lewis X
(Gal.beta.1-4(Fuc.alpha.1-- 3)(SO.sub.4-6)GlcNAc) was generated
according to the method described by Kohler and Milstein (Nature
256,495-497(1975)), and subsequently used to produce
anti-carbohydrate antibodies (Kannagi, R., and Hakomori, S.(1986)
in Handbook of Experimental Immunology, Vol. 4, Applications of
immunological methods in biomedical sciences (Weir, D. M., H
erzenberg, L., Blackwell, C., and Herzenberg, L. A., eds) pp. 117
.1-117.20, Blackwell Scienctific Pub. Inc., Boston). Briefly,
6-sulfo Lewis X ceramide (Gal.beta.1-4(Fuc.alpha.1-3)
(SO.sub.4-6)GlcNAc.beta.1-3Gal.beta- .1-4Glc.beta.1-Cer) was
adsorbed to Salmonella minnesota R595 strain and used for repeated
intraperitoneal immunization of BALB/c mice on day 0(5 .mu.g
glycolipid), day 3(10 .mu.g), day 7(15 .mu.g), day 12(20 .mu.g),
day 17(25 .mu.g) and day 31(35 .mu.g). Three days after the final
immunization, the spleen cells were harvested and fused with mouse
myeloma p3/X63-Ag8U1. The same glycolipid was used as the antigen
in ELISA as used for hybridoma culture supernatants for the cloning
procedures. ELISA was performed using glycolipid antigens
immobilized at the bottom of 96-well culture plates by the standard
method (Hakomori, S., and Kannagi, R., (1986) in Handbook of
Experimental Immunology, Vol. 1, Immunochemistry (Weir, D. M.,
Herzenberg, L., Blackwell, C., and Herzenberg, L. A., eds) pp.
9.1-9.39, Blackwell Scientific Pub. Inc., Boston).
Peroxidase-conjugated goat anti-mouse IgM (.mu.-chain specific,
Cappel Inc.) was used as the second antibody.
[0213] A hybridoma cell line G72, which secreted murine IgM
monoclonal antibody (G72) reacting with 6-sulfo sialyl
N-acetyllactosamine (NeuAc.alpha.2-3Gal.beta.1-4 (SO.sub.4-6)
GlcNAc) structure was similarly prepared using 6-sulfo sialyl Lewis
X ceramide.
[0214] Mouse IgM monoclonal antibody (G152) that reacts with
6-sulfated sialyl Lewis X ceramide but does not react with
6'-sulfated sialyl Lewis X ceramide, 6,6'-bis-sulfated sialyl Lewis
X ceramide, 6-sulfated Lewis X ceramide, Lewis X ceramide, or the
like (thus recognizes 6-sulfated sialyl Lewis X antigen) was
prepared using 6-sulfated sialyl Lewis X ceramide as an antigen by
the method described in J. Biol. Chem. 273, 11225-11233 (1998).
[0215] CSLEX-1 monoclonal antibody (Cancer Res. 44, 5279-5285
(1984)); reacting with sialyl Lewis X antigen) was used to detect
sialyl Lewis X antigen.
[0216] Cell-surface expression of antigenic epitopes was surveyed
by FACS as described in (Biochem. Biophys. Res. Commun., 230,
546-551 (1997)) using a FACScan (Becton Dickinson Co.).
[0217] (10) Northern and Genomic Southern Blot Analyses
[0218] (10-1) Mouse
[0219] Total RNA (20 .mu.g) was prepared from C57 BL/6J mouse
tissues as described (Anal. Biochem. 162, 156-159 (1987)). Genomic
DNA (10 .mu.g) prepared from murine D3 embryonic stem cells (J.
Embryol. Exp. Morphol., 87, 27-45 (1985)) was digested for 4 h with
appropriate restriction enzymes. The radioactive probe was the same
as that used for screenig of the mouse day-7 embryo cDNA library.
The blots were washed at 55.degree. C. in 2.times. SSPE, 0.1% SDS,
and finally in 0.1.times. SSPE, 0.1% SDS at 55.degree. C. The
membranes were exposed to a BAS-imaging plate and then the
radioactivity on the membrane was determined with a BAS 2000
radioimage analyzer (Fuji Film Co.).
[0220] (10-2) Human
[0221] Total RNA was prepared from human tissues in the same manner
as in (10-1) above. Bpu1102 I-BamHI 368 bp fragment (nucleotide
numbers 910 to 1277 in SEQ ID NO: 3) of the DNA of the present
invention derived from human was used as a probe. Blotting was
carried out in the same manner as in (10-1) above.
[0222] (11) In situ Hybridization
[0223] Specimens from C57 BL/6J mice were subjected to
hematoxylin-eosin staining or in situ hybridization. As the probe
for the polypeptide of the present invention, a 0.6 kbp Pst I
fragment of the cDNA (nucleotide numbers 962 to 1561 in SEQ ID
NO:1) was subcloned into pBluescript II SK-. Sense and antisense
cRNA probes were prepared by in vitro transcription with a DIG RNA
labeling kit (Boehringer Mannheim Co., Germany).
[0224] (12) Fluorescence in situ Hybridization Analysis
[0225] Fluorescence in situ hybridization (FISH) analysis was
carried out in accordance with the method described in Genomics,
17, 514-515 (1993) using the DNA of the present invention derived
from human (nucleotide numbers 1 to 2409 in SEQ ID NO: 3) as a
probe.
[0226] <2> RESULTS
[0227] (1) Cloning of the DNA of the Present Invention
[0228] Mouse chondroitin 6-sulfotransferase had cloned previously.
By searching in the EST database, we found a small sequence with
similarity to the catalystic portion of mouse chondroitin
6-sulfotransferase (Genbank accession number AA103962). We obtained
the corresponding cDNA fragment by RT-PCR (nucleotide numbers 1139
to 1506 in SEQ ID NO:1). Approximately 8.times.10.sup.5 plaques of
a mouse day-7 embryo cDNA library were screened using the cDNA
fragment as a probe, and six independent clones were obtained. The
nucleotide sequence of the largest cDNA insert (2.2 kb) was
determined (SEQ ID NO:1). The determined 2150-bp cDNA had a single
open reading frame consisting of 483 amino acids, with a molecular
mass of 52829 Da and four potential N-linked glycosylation sites
(SEQ ID NO:1). The sequence around the first ATG codon fitted
Kozak's rule (Cell, 44, 283-292 (1986)), and the upstream region
contained an in-frame stop codon. Hydropathy plot analysis
indicated the presence of one prominent hydrophobic segment 20
residues in length in the amino-terminal region
(Ala.sup.8-Leu.sup.27) predicting that the polypeptide of the
present invention is type II transmembrane protein (FIG. 1). The
polypeptide of the present invention showed 25% and 27% homology
with mouse chondoroitin 6-sulfotransferase and human keratan
sulfate Gal-6-sulfotransferase, respectively. However, no
significant homology in amino acid sequence was observed between
the protein and other known sulfotransferases (J. Biol. Chem., 267,
15744-15750 (1992), J. Biol. Chem., 272, 13980-13985 (1997), J.
Biol. Chem., 272, 28008-28019 (1997), J. Biol. Chem., 272,
29942-29946 (1997), J. Biol. Chem., 272, 4864-4868 (1997)).
[0229] The DNA of the present invention derived from human (SEQ ID
NO: 3) was obtained by screening the .lambda.gt11 library
containing cDNA of human fetal brain using the DNA represented by
SEQ ID NO: 1. The obtained DNA consisted of 2409 bp and contained a
single open reading frame consisting of 484 amino acid residues
(SEQ ID NO: 3). Hydropathy plot analysis suggested that this
polypeptide is type II transmembrane protein having transmembrane
domain at the amino terminal region.
[0230] The sequence in the vicinity of the first ATG codon was in
agreement with the Kozak's rule (Cell, 44, 283-292 (1986)).
[0231] Another ATG codon exists 41 nucleotide numbers upstream from
the first ATG codon. The sequence in the vicinity of this ATG codon
was also in agreement with the Kozak's rule. This ATG codon was
also found in the corresponding position in the DNA of the present
invention derived from mouse. There is no termination codon between
the first ATG codon and this ATG codon in the human nor the mouse
DNA. Therefore, both of the above two ATG codons can function as an
initiation codon in the DNA of the present invention.
[0232] The amino acid sequence of the polypeptide encoded by the
DNA of SEQ ID NO: 1 and the amino acid sequence of the polypeptide
encoded by the DNA of SEQ ID NO: 3 are shown by SEQ ID NO: 2 and
SEQ ID NO: 4, respectively. Homology between SEQ ID NO: 2 and SEQ
ID NO: 4 was 85% or more.
[0233] (2) Expression of the Polypeptide of the Present Invention
from the DNA of the Present Invention
[0234] The DNA of the present invention, from which the bulk of the
5'- and 3'-non-coding regions was removed (nucleotide numbers 467
to 1921 in SEQ ID NO:1) was inserted into a mammalian expression
vector pcDNA3 and overexpressed in COS-7 cells. Extracts of the
transfected cells were assayed for sulfotransferase activity using
.sup.35S-labeled PAPS as the sulfate group donor and various
glycoconjutages as sulfate group acceptors: chondroitin,
chondroitin 4-sulfate, chondroitin 6-sulfate, dermantan sulfate,
keratan sulfate, desulfated keratan sulfate, CDSNS-heparin, mucin
from porcine stomach and mucin from bovine submaxillary gland did
not serve as acceptors. We examined
GlcNAc.beta.1-3Gal.beta.1-4GlcNAc as a sulfate group acceptor.
Superdex 30 chromatography of the reaction mixture indeed revealed
a radioactive peak in cells transfected with a vector
(pcDNA3-GlcNAc6ST) containig the cDNA of the correct orientation
(sense DNA) slighly larger than the acceptor, indicating that the
acceptor was sulfated (FIG. 2A). The extract from untransfected
cells or those transfected with a vector (pcDNA3-GlcNAc6STA)
containing the cDNA of the reverse orientation (the antisense cDNA)
showed much less sulfotransferase activity (Table 1). The acitivity
was calculated from the radioactivity contained in Fraction Number
85 to 89 in Superdex 30 chromatography (FIG. 2A). Values are shown
that values obtained in the absence of the acceptor were subtracted
from the value obtained in the presence of the acceptor. Values of
averages .+-.S.D. of triplicate culture are shown.
1 TABLE 1 Sulfotransferase activity Vector pmole/hour/mg protein
None (untransfected) 1.1 .+-. 0.7 Sense cDNA 10.6 .+-. 2.4
Antisense cDNA 1.8 .+-. 0.9
[0235] In contrast, Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4GlcNAc
did not serve as an acceptor (FIG. 2B). Therefore, it was confirmed
that the polypeptide of the present invention transferred sulfate
group to GlcNAc.beta.1-3Gal.beta.1-4GlcNAc, but didn't to
Gal.beta.1-4GlcNAc.beta.- 1-3Gal.beta.1-4GlcNAc.
[0236] COS-7 cells were transfected with the DNA of the present
invention from which the bulk of 5'- and 3'-non-coding region was
removed (nucleotide numbers 387 to 1844 in SEQ ID NO:3) in the same
manner as described above and the extracts of resulting
transfectants were examined for its sulfotransferase activity
(using GlcNAc.beta.1-3Gal.beta.1-4GlcNA-
c.beta.1-3Gal.beta.1-4GlcNAc as a sulfate group acceptor) in the
same manner as described above. As a result, the cells transfected
with the vector (pcDNA3-hGlcNAc6ST) containing the cDNA with the
correct orientation (sense cDNA) exhibited fivefold or more as high
sulfotransferase activity as untransfected cells or the cells
transfected with the vector (pcDNA3-hGlcNAc6STA) containing the
cDNA with the reverse orientation (antisense cDNA)(Table 2). The
activity was calculated from radioactivity of fractions obtained by
Superdex 30 chromatography. Values were shown that values obtained
in the absence of the acceptor were subtracted from the value
obtained in the presence of the acceptor. Values of averages
.+-.S.D. of triplicate culture are shown.
2 TABLE 2 Sulfotransferase activity Vector pmole/hour/mg protein
None (untransfected) 1.33 .+-. 0.62 Sense cDNA 6.67 .+-. 0.51
Antisense cDNA 1.29 .+-. 0.23
[0237] (3) Determination of the Position Where a Sulfate Group is
Transferred by the Polypeptide of the Present Invention (Method for
Measuring the Activity of the Polypeptide of the Present
Invention)
[0238] To determine the position to which .sup.35SO.sub.4 was
transferred to GlcNAc.beta.1-3Gal.beta.1-4GlcNAc, we degraded the
radioactive product by a reatction sequence of N-deacetylation,
deamination, and NaBH.sub.4 reduction. After degradation, two
radioactive products were detected on the paper chromatogram (FIG.
3A). The faster moving peak migrated to the position of
(SO.sub.4-6)2,5-anhydromannitol. The slower moving peak was thought
to be undegraded materials due to incomplete deacetylation because
the proportion of the slower moving peak was decreased when
.sup.35S-labeled GlcNAc.beta.1-3Gal.beta.1-4GlcNAc was subjected to
prolonged hydrazinolysis reaction. When the faster migrating
material was analyzed by HPLC, .sup.35S-radioactivity was co-eluted
with (SO.sub.4-6).sup.3H-2,5-anhydromannitol (FIG. 3B). The
.sup.35S- and .sup.3H-radioactivities also co-migrated upon TLC,
using a solvent system which can separate 6-sulfo
2,5-anhydromannnitol from 4-sulfo or 3-sulfo 2,5-anhydromannitol
(Biochem. J., 319,209-216(19 96)). These results indicate that
.sup.35SO.sub.4 was transferred to position 6 of GlcNAc residue
located at the non-reducing end of GlcNAc.beta.1-3Gal.beta.1-4Glc-
NAc. Therefore, the cloned DNA of the present invention was found
to encode the polypeptide of the present invention.
[0239] (4) Expression of Sulfated Sugar by Transfecting the DNA of
the Present Invention into Cells
[0240] Expression of a new antigenic epitope was examined in COS-7
cells transfected with the DNA of the present invention derived
from mouse. For analysis of expression of sulfated
sialyl-N-acetyllactosamine structure, we used G72 antibody. It
reacted with 6-sulfo sialyl Lewis X ceramide as well as SL2L4, but
not with 6'-sulfo sialyl Lewis X ceramide or sialyl-paraglobosides
(FIG. 4), indicating that the minimum structure required for the
reactivity of the antibody was NeuAc.alpha.2-3Gal.beta.1-
-4(SO.sub.4-6)GlcNAc. COS-7 cells transfected with the DNA of the
present invention expressed the G72 antigen (FIG. 5B). The
untransfected cells (FIG. 5A) or the cells transfected with the
antisense cDNA (FIG. 5C) were not reactive with the antibody.
Neuramimidase digestion abolished the antigenicity (FIG. 5E). Thus,
we reached to a conclusion that the polypeptide of the present
invention is involved in synthesis of
NeuAc.alpha.2-3Gal.beta.1-4(SO.sub.4-6)GlcNAc antigen.
[0241] In order to obtain further evidence that the polypeptide of
the present invention is involeved in formation of 6-sulfo
N-acetyllactosamine structure, we doubly transfected COS-7 cells
with the DNA of the present invention derived from mouse and the
cDNA of fucosyltransferase IV. The latter enzyme is known to be
able to form Lewis X structure (Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc)
by transferring fucose to N-acetyllactosamine (J. Biol. Chem., 266,
17467-17477 (1991), Cell, 63, 1349-1356 (1990)). To monitor the
cell surface change due to transfection, we used monoclonal
antibody AG223, which specifically reacted with 6-sulfo Lewis X
structure (Gal.beta.1-4(Fuc.alpha.1-3)(SO.su- b.4-6)GlcNAc), and
did not react with Lewis X (Gal.beta.1-4(Fuc.alpha.1-3)- GlcNAc)
nor with 61-sulfo Lewis X structure ((SO.sub.4-6)Gal.beta.1-4(Fuc.-
alpha.1-3) GlcNAc)(FIG. 4). Transfection with the DNA of the
present invention and the cDNA of fucosyltransferase indeed yielded
cells positive for the 6-sulfo Lewis X antigen (FIG. 6C), but cells
transfected only with one cDNA did not (FIGS. 6A, B). When the
cells were transfected with only the cDNA of fucosyltransferase,
the cells became positive for Lewis X antigen (FIG. 6D)
Furthermore, expression of new antigenic epitope in COS-7 cells
transfected with the DNA of the present invention derived from
human was examined.
[0242] Expression of sulfated sugar on the cell surface was
examined in the same manner as described above using the CSLEX-1
antibody (Cancer Res., 44, 5279-5285 (1984); reacting with sialyl
Lewis X antigen), the G72 antibody, and the G152 antibody (which
reacted with 6-sulfated sialyl Lewis X ceramide but did not react
with 6'-sulfated sialyl Lewis X ceramide, 6,6'-bis-sulfated sialyl
Lewis X ceramide, 6-sulfated Lewis X ceramide, Lewis X ceramide, or
the like). The results are shown in FIG. 7.
[0243] As a result, untransfected cells did not react with any of
the antibodies. The cells transfected with cDNA of
fucosyltransferase alone reacted with only CELEX-1 antibody. The
cells transfected with the DNA of the present invention derived
from human reacted with only the G72 antibody. The cells
cotransfected with the DNA of the present invention derived from
human and the cDNA of fucosyltransferase reacted with any of the
CSLEX-1 antibody, the G72 antibody, and the G152 antibody.
[0244] These results indicated that sulfated sugar having
6-sulfated sialyl-N-acetyllactosamine structure could be produced
by transfecting the DNA of the present invention into cells and
that sulfated sugar having 6-sulfated sialyl Lewis X structure
could be produced by concurrently transfecting the DNA of the
present invention and cDNA of fucosyltransferase into cells.
[0245] (5) Nothern Blot and Southern Blot Analyses
[0246] The result of the examination using the adult mouse organs
(cerebellum, cerebrum, eyeball, heart, lung, muscle, spleen,
thymus, liver, pancreas, kidney, stomach, intestine, uterus, ovary,
and testis) indicated that the polypeptide of the present invention
was strongly expressed in the cerebrum, cerebellum, eye, lung and
pancreas (FIG. 8A). Moderate signal was also detected in the
intestine, uterus, ovary, mesentric lymph nodes and peripheral
(FIG. 8A). The size of major transcript was 3.9 kb. On southern
blots, a single band was reacted with the probe after digestion
with EcoRI, RcoRV or SacI, indicating that the polypeptide of the
present invention was a single copy gene (FIG. 8B).
[0247] As a result of examining human tissues (heart, brain,
placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,
thymus, prostate, testis, ovary, intestine, colon, peripheral blood
lymphocytes, stomach, thyroid gland, spinal cord, lymph node,
trachea, adrenal body, and bone marrow), the polypeptide of the
present invention was strongly expressed in bone marrow, peripheral
blood lymphocytes, spleen, brain, spinal cord, ovary, and placenta.
Moderate signal was observed in lymph node, thymus, heart, lung,
trachea, stomach, intestine, colon, thyroid gland, prostate, and
adrenal body. The size of major transcription products was 3.6
kb.
[0248] (6) In situ Hybridization Analyses
[0249] In situ hybridization was performed to determine the
expression sites of the polypeptide of the present invention. In
the adult mouse brain, strong signals were detected in pyramidal
cells in the CA3 subregion of the hippocampus, cerebellar nucleus
and Purkinje cells. Moderate signals were detected in the other
subregions including CA1 subregion of the hippocampus, thalamus,
pontine nucleus, olfactory tubercle and olfactory bulb.
[0250] Mesenteric lymph nodes were used to disclose the
localization of the DNA transcripts in lymphoid tissues. HEV (high
endothelial venules), where the ligand for L-selectin is located
(J. Cell Biol., 113, 1213-1221 (1991)), are present in the
paracortex. They have a cuboidal endothelium with fairly large oval
nuclei and a few cytoplasm. The polypeptide of the present
invention was specifically expressed on these endothelium.
[0251] (7) Fluorescence in situ Hybridization Analysis
[0252] Fluorescence in situ hybridization analysis was carried out
to determine the position of the DNA of the present invention
derived from human on the chromosome. As a result, the DNA of the
present invention derived from human was found to locate at the
chromosome 7q31 site.
Sequence CWU 1
1
10 1 2150 DNA Mus musculus CDS (470)...(1918) 1 ggctagggca
gcggagtctc gcggctccct cgaaggcttg gggaccccta gcagaagaga 60
accggagaga aaccgaggag agtgctagcc ggacagtccg ccggtcgggg atctggggac
120 gctccgaggc gcaccctccg ctccaggtcc ttctcggagc cgctgccatg
ggagagccag 180 ccctgggcgc cggggaccag cagcctctgc cgccgcgccc
gcctcggatc ggcggcccca 240 gtcccggcgc ccgcagccgg cctgcagcgt
ccccctcctg ggctgcaggg ccgcctccgc 300 cgcgccgccg gccccggctg
tgcctgtgat gagccgcagc tcgccgcgag ctctgccccc 360 cggtgcgctt
ccccggccgc tgccggccgc gcctgccgcc gtgcagcggg ccctgctccc 420
gccgtggccc cggcgcgcag gacgccgctg gcctgcgtcc ccgctcggg atg aag gta
478 Met Lys Val 1 ttt cgc agg aag gcg ctg gtg ctg tgc gcg ggc tat
gca ctg cta ctg 526 Phe Arg Arg Lys Ala Leu Val Leu Cys Ala Gly Tyr
Ala Leu Leu Leu 5 10 15 gtg ctc acg atg ctc aac ctc ttg gac tac aag
tgg cat aaa gag ccg 574 Val Leu Thr Met Leu Asn Leu Leu Asp Tyr Lys
Trp His Lys Glu Pro 20 25 30 35 ctg cag cag tgc aac ccc gac ggg cct
ctg ggt gcc gcg gta ggg gcg 622 Leu Gln Gln Cys Asn Pro Asp Gly Pro
Leu Gly Ala Ala Val Gly Ala 40 45 50 gcc ggg gcc ggc tgg gga cgg
ccg ggg tcg cct cct gca gcg cca ccc 670 Ala Gly Ala Gly Trp Gly Arg
Pro Gly Ser Pro Pro Ala Ala Pro Pro 55 60 65 cgc gct cac tct cgc
atg gac ccc cgc acc ccg tac cgc cct cct gcc 718 Arg Ala His Ser Arg
Met Asp Pro Arg Thr Pro Tyr Arg Pro Pro Ala 70 75 80 gcg ggc gtg
ggg gca gtt ccc gca gcc gcg gct ggg agt gca gga gct 766 Ala Gly Val
Gly Ala Val Pro Ala Ala Ala Ala Gly Ser Ala Gly Ala 85 90 95 gcg
gcc tct ctg ggc aat gct act cga ggc acc agg ggt gga ggg gac 814 Ala
Ala Ser Leu Gly Asn Ala Thr Arg Gly Thr Arg Gly Gly Gly Asp 100 105
110 115 aag cgg cag ttg gtg tat gtg ttc acc acg tgg cgc tcg ggc tcg
tcc 862 Lys Arg Gln Leu Val Tyr Val Phe Thr Thr Trp Arg Ser Gly Ser
Ser 120 125 130 ttc ttc ggt gag ctc ttc aac cag aac cct gag gtg ttc
ttc ctc tat 910 Phe Phe Gly Glu Leu Phe Asn Gln Asn Pro Glu Val Phe
Phe Leu Tyr 135 140 145 gag cct gtg tgg cac gtg tgg caa aaa ctg tac
ccc ggg gac gcc gtt 958 Glu Pro Val Trp His Val Trp Gln Lys Leu Tyr
Pro Gly Asp Ala Val 150 155 160 tcc ctg cag ggg gca gcg cgg gac atg
ctg agc gct ctc tac cgc tgc 1006 Ser Leu Gln Gly Ala Ala Arg Asp
Met Leu Ser Ala Leu Tyr Arg Cys 165 170 175 gat ctt tcg gtt ttc cag
ctg tat agc ccc gca ggc agt ggg ggg cgc 1054 Asp Leu Ser Val Phe
Gln Leu Tyr Ser Pro Ala Gly Ser Gly Gly Arg 180 185 190 195 aac ctc
acc act ctg ggc atc ttt ggg gca gcc act aac aag gtg gta 1102 Asn
Leu Thr Thr Leu Gly Ile Phe Gly Ala Ala Thr Asn Lys Val Val 200 205
210 tgc tcc tcg cca ctc tgt cct gcc tac cgc aag gag gtc gtc gga ctg
1150 Cys Ser Ser Pro Leu Cys Pro Ala Tyr Arg Lys Glu Val Val Gly
Leu 215 220 225 gtg gac gac cgc gtg tgc aaa aag tgc cca cct caa cgc
ctg gca cgc 1198 Val Asp Asp Arg Val Cys Lys Lys Cys Pro Pro Gln
Arg Leu Ala Arg 230 235 240 ttc gag gag gag tgt cgc aag tac cgc acg
gtg gtt atc aag ggc gtg 1246 Phe Glu Glu Glu Cys Arg Lys Tyr Arg
Thr Val Val Ile Lys Gly Val 245 250 255 cgg gtc ttc gat gtg gct gtg
ttg gcg ccg ctg ctt aaa gat cca gcc 1294 Arg Val Phe Asp Val Ala
Val Leu Ala Pro Leu Leu Lys Asp Pro Ala 260 265 270 275 ttg gac ctc
aag gtc atc cac cta gta cgt gat cct cgt gct gtt gcc 1342 Leu Asp
Leu Lys Val Ile His Leu Val Arg Asp Pro Arg Ala Val Ala 280 285 290
agc tcc cgc atc cgc tcg cgt cac ggc ctc atc cgg gaa agc cta cag
1390 Ser Ser Arg Ile Arg Ser Arg His Gly Leu Ile Arg Glu Ser Leu
Gln 295 300 305 gtg gtg cga agc cgg gat cca aga gcc cac cgc atg ccc
ttc ctg gag 1438 Val Val Arg Ser Arg Asp Pro Arg Ala His Arg Met
Pro Phe Leu Glu 310 315 320 gct gct ggc cac aag ctt ggt gcc aag aag
gag ggt atg ggt ggc cca 1486 Ala Ala Gly His Lys Leu Gly Ala Lys
Lys Glu Gly Met Gly Gly Pro 325 330 335 gca gac tac cac gct ctg ggt
gca atg gag gtc atc tgc aac agt atg 1534 Ala Asp Tyr His Ala Leu
Gly Ala Met Glu Val Ile Cys Asn Ser Met 340 345 350 355 gcc aag acg
ctg caa aca gcc ctg cag cct cct gac tgg ctg cag gga 1582 Ala Lys
Thr Leu Gln Thr Ala Leu Gln Pro Pro Asp Trp Leu Gln Gly 360 365 370
cac tac ttg gtg gtg agg tac gag gat ctg gtg gga gac ccc gtt aag
1630 His Tyr Leu Val Val Arg Tyr Glu Asp Leu Val Gly Asp Pro Val
Lys 375 380 385 acc cta cgg agg gta tat gac ttt gtg ggg ctg ctg gtg
agt ccc gaa 1678 Thr Leu Arg Arg Val Tyr Asp Phe Val Gly Leu Leu
Val Ser Pro Glu 390 395 400 atg gag cag ttt gcc ctg aac atg acc agt
ggt tcg ggc tcc tcc tcc 1726 Met Glu Gln Phe Ala Leu Asn Met Thr
Ser Gly Ser Gly Ser Ser Ser 405 410 415 aag cct ttc gtg gtg tca gct
cgc aat gcc act cag gcc gcc aat gcc 1774 Lys Pro Phe Val Val Ser
Ala Arg Asn Ala Thr Gln Ala Ala Asn Ala 420 425 430 435 tgg cgg acc
gcg ctc acc ttc cag cag atc aaa cag gtg gag gag ttt 1822 Trp Arg
Thr Ala Leu Thr Phe Gln Gln Ile Lys Gln Val Glu Glu Phe 440 445 450
tgc tac cag ccc atg gcc gtg ctg ggc tat gag cgg gtt aac agt cct
1870 Cys Tyr Gln Pro Met Ala Val Leu Gly Tyr Glu Arg Val Asn Ser
Pro 455 460 465 gag gag gtc aaa gac ctc agc aag acc ttg ctc agg aag
ccc cgg ctt 1918 Glu Glu Val Lys Asp Leu Ser Lys Thr Leu Leu Arg
Lys Pro Arg Leu 470 475 480 tgagaagggt tcccaagaga tctgacactc
tccggagaca cccacaaaaa ggatggtgtt 1978 gtgtttaaac aaacacagcc
cagacccaag ctgaggaagc ccacatattc tattatagat 2038 atataatata
aataaccaca caggcacttg ctgtcaacgt tttgagtcag tgcatttcaa 2098
ggaacagccc tcaactcaca cgacaaactt ctggccctcc aacaagacac ac 2150 2
483 PRT Mus musculus 2 Met Lys Val Phe Arg Arg Lys Ala Leu Val Leu
Cys Ala Gly Tyr Ala 1 5 10 15 Leu Leu Leu Val Leu Thr Met Leu Asn
Leu Leu Asp Tyr Lys Trp His 20 25 30 Lys Glu Pro Leu Gln Gln Cys
Asn Pro Asp Gly Pro Leu Gly Ala Ala 35 40 45 Val Gly Ala Ala Gly
Ala Gly Trp Gly Arg Pro Gly Ser Pro Pro Ala 50 55 60 Ala Pro Pro
Arg Ala His Ser Arg Met Asp Pro Arg Thr Pro Tyr Arg 65 70 75 80 Pro
Pro Ala Ala Gly Val Gly Ala Val Pro Ala Ala Ala Ala Gly Ser 85 90
95 Ala Gly Ala Ala Ala Ser Leu Gly Asn Ala Thr Arg Gly Thr Arg Gly
100 105 110 Gly Gly Asp Lys Arg Gln Leu Val Tyr Val Phe Thr Thr Trp
Arg Ser 115 120 125 Gly Ser Ser Phe Phe Gly Glu Leu Phe Asn Gln Asn
Pro Glu Val Phe 130 135 140 Phe Leu Tyr Glu Pro Val Trp His Val Trp
Gln Lys Leu Tyr Pro Gly 145 150 155 160 Asp Ala Val Ser Leu Gln Gly
Ala Ala Arg Asp Met Leu Ser Ala Leu 165 170 175 Tyr Arg Cys Asp Leu
Ser Val Phe Gln Leu Tyr Ser Pro Ala Gly Ser 180 185 190 Gly Gly Arg
Asn Leu Thr Thr Leu Gly Ile Phe Gly Ala Ala Thr Asn 195 200 205 Lys
Val Val Cys Ser Ser Pro Leu Cys Pro Ala Tyr Arg Lys Glu Val 210 215
220 Val Gly Leu Val Asp Asp Arg Val Cys Lys Lys Cys Pro Pro Gln Arg
225 230 235 240 Leu Ala Arg Phe Glu Glu Glu Cys Arg Lys Tyr Arg Thr
Val Val Ile 245 250 255 Lys Gly Val Arg Val Phe Asp Val Ala Val Leu
Ala Pro Leu Leu Lys 260 265 270 Asp Pro Ala Leu Asp Leu Lys Val Ile
His Leu Val Arg Asp Pro Arg 275 280 285 Ala Val Ala Ser Ser Arg Ile
Arg Ser Arg His Gly Leu Ile Arg Glu 290 295 300 Ser Leu Gln Val Val
Arg Ser Arg Asp Pro Arg Ala His Arg Met Pro 305 310 315 320 Phe Leu
Glu Ala Ala Gly His Lys Leu Gly Ala Lys Lys Glu Gly Met 325 330 335
Gly Gly Pro Ala Asp Tyr His Ala Leu Gly Ala Met Glu Val Ile Cys 340
345 350 Asn Ser Met Ala Lys Thr Leu Gln Thr Ala Leu Gln Pro Pro Asp
Trp 355 360 365 Leu Gln Gly His Tyr Leu Val Val Arg Tyr Glu Asp Leu
Val Gly Asp 370 375 380 Pro Val Lys Thr Leu Arg Arg Val Tyr Asp Phe
Val Gly Leu Leu Val 385 390 395 400 Ser Pro Glu Met Glu Gln Phe Ala
Leu Asn Met Thr Ser Gly Ser Gly 405 410 415 Ser Ser Ser Lys Pro Phe
Val Val Ser Ala Arg Asn Ala Thr Gln Ala 420 425 430 Ala Asn Ala Trp
Arg Thr Ala Leu Thr Phe Gln Gln Ile Lys Gln Val 435 440 445 Glu Glu
Phe Cys Tyr Gln Pro Met Ala Val Leu Gly Tyr Glu Arg Val 450 455 460
Asn Ser Pro Glu Glu Val Lys Asp Leu Ser Lys Thr Leu Leu Arg Lys 465
470 475 480 Pro Arg Leu 3 2409 DNA Homo sapiens CDS (390)...(1841)
3 cgaggaacga gtgacagccg gacagtccgc cgggcggtga tccggggccg ctcccgggcg
60 cgccctcggc tccaggtcct acccggagcc gctgccatgg gagagccagc
cttgggcgct 120 ggggaccagc cgccgcgccc gcctcggagt cgcggcccga
gtcccggcgc cagcagccag 180 cccgctgcgt ccccttcccg ggctgcaggg
ctgcctccgc cgcgccgccg gcccggattg 240 tgcctgtgat gagccgcagc
ccgcagcgag ctctgccccc gggcgcgctc cctcggctgc 300 tccaggctgc
gcctgcagcg cagccgcgtg ccctgctccc gcagtggccc cggcgcccag 360
gacgccgctg gcccgcgtcc cctctcgga atg aag gtg ttc cgt agg aag gcg 413
Met Lys Val Phe Arg Arg Lys Ala 1 5 ctg gtg ttg tgc gcg ggc tat gca
ctg ctg ctg gtg ctc act atg ctc 461 Leu Val Leu Cys Ala Gly Tyr Ala
Leu Leu Leu Val Leu Thr Met Leu 10 15 20 aac ctc ctg gac tac aag
tgg cac aag gag ccg ctg cag cag tgc aac 509 Asn Leu Leu Asp Tyr Lys
Trp His Lys Glu Pro Leu Gln Gln Cys Asn 25 30 35 40 ccc gat ggg ccg
ctg ggt gcc gca gcg ggg gca gcc gga ggc aag ctg 557 Pro Asp Gly Pro
Leu Gly Ala Ala Ala Gly Ala Ala Gly Gly Lys Leu 45 50 55 ggg gcg
ccc agg gcc gcc tcc ggc cgg gcc gcc ccg tgc tca tgc ccg 605 Gly Ala
Pro Arg Ala Ala Ser Gly Arg Ala Ala Pro Cys Ser Cys Pro 60 65 70
ttt gga cct ccg cac tcc tta ccg ccc tcc cgc tgc cgc cgt cgg ggc 653
Phe Gly Pro Pro His Ser Leu Pro Pro Ser Arg Cys Arg Arg Arg Gly 75
80 85 gat act ctg cag ccg cgg cag gga tgg cgg ggg ttg cgg ccc ctc
cag 701 Asp Thr Leu Gln Pro Arg Gln Gly Trp Arg Gly Leu Arg Pro Leu
Gln 90 95 100 gca atg gca ctc ggg gca ccg gag ggc gtc ggg gac aag
cgg cac tgg 749 Ala Met Ala Leu Gly Ala Pro Glu Gly Val Gly Asp Lys
Arg His Trp 105 110 115 120 atg tac gtg ttc acc acg tgg cgc tct ggc
tcg tcg ttc ttc ggc gag 797 Met Tyr Val Phe Thr Thr Trp Arg Ser Gly
Ser Ser Phe Phe Gly Glu 125 130 135 cta ttc aac cag aat ccc gag gtg
ttc ttt ctc tac gag cca gtg tgg 845 Leu Phe Asn Gln Asn Pro Glu Val
Phe Phe Leu Tyr Glu Pro Val Trp 140 145 150 cat gta tgg caa aaa ctg
tat ccg ggg gac gcc gtt tcc ctg cag ggg 893 His Val Trp Gln Lys Leu
Tyr Pro Gly Asp Ala Val Ser Leu Gln Gly 155 160 165 gca gcg cgg gac
atg ctg agc gct ctt tac cgc tgc gac ctc tct gtc 941 Ala Ala Arg Asp
Met Leu Ser Ala Leu Tyr Arg Cys Asp Leu Ser Val 170 175 180 ttc cag
ttg tat agc ccc gcg ggc agc ggg ggg cgc aac ctc acc acg 989 Phe Gln
Leu Tyr Ser Pro Ala Gly Ser Gly Gly Arg Asn Leu Thr Thr 185 190 195
200 ctg ggc atc ttc ggc gca gcc acc aac aag gtg gtg tgc tcg tca cca
1037 Leu Gly Ile Phe Gly Ala Ala Thr Asn Lys Val Val Cys Ser Ser
Pro 205 210 215 ctc tgc ccc gcc tac cgc aag gag gtc gtg ggg ttg gtg
gac gac cgc 1085 Leu Cys Pro Ala Tyr Arg Lys Glu Val Val Gly Leu
Val Asp Asp Arg 220 225 230 gtg tgc aag aag tgc ccg cca cag cgc ctg
gcg cgt ttc gag gag gag 1133 Val Cys Lys Lys Cys Pro Pro Gln Arg
Leu Ala Arg Phe Glu Glu Glu 235 240 245 tgc cgc aag tac cgc aca cta
gtc ata aag ggt gtg cgc gtc ttc gac 1181 Cys Arg Lys Tyr Arg Thr
Leu Val Ile Lys Gly Val Arg Val Phe Asp 250 255 260 gtg gcg gtc ttg
gcg cca ctg ctg cga gac ccg gcc ctg gac ctc aag 1229 Val Ala Val
Leu Ala Pro Leu Leu Arg Asp Pro Ala Leu Asp Leu Lys 265 270 275 280
gtc atc cac ttg gtg cgt gat ccc cgc gcg gtg gcg agt tca cgg atc
1277 Val Ile His Leu Val Arg Asp Pro Arg Ala Val Ala Ser Ser Arg
Ile 285 290 295 cgc tcg cgc cac ggc ctc atc cgt gag agc cta cag gtg
gtg cgc agc 1325 Arg Ser Arg His Gly Leu Ile Arg Glu Ser Leu Gln
Val Val Arg Ser 300 305 310 cga gac ccg cga gct cac cgc atg ccc ttc
ttg gag gcc gcg ggc cac 1373 Arg Asp Pro Arg Ala His Arg Met Pro
Phe Leu Glu Ala Ala Gly His 315 320 325 aag ctt ggc gcc aag aag gag
ggc gtg ggc ggc ccc gca gac tac cac 1421 Lys Leu Gly Ala Lys Lys
Glu Gly Val Gly Gly Pro Ala Asp Tyr His 330 335 340 gct ctg ggc gct
atg gag gtc atc tgc aat agt atg gct aag acg ctg 1469 Ala Leu Gly
Ala Met Glu Val Ile Cys Asn Ser Met Ala Lys Thr Leu 345 350 355 360
cag aca gcc ctg cag ccc cct gac tgg ctg cag ggc cac tac ctg gtg
1517 Gln Thr Ala Leu Gln Pro Pro Asp Trp Leu Gln Gly His Tyr Leu
Val 365 370 375 gtg cgg tac gag gac ctg gtg gga gac ccc gtc aag aca
cta cgg aga 1565 Val Arg Tyr Glu Asp Leu Val Gly Asp Pro Val Lys
Thr Leu Arg Arg 380 385 390 gtg tac gat ttt gtg gga ctg ttg gtg agc
ccc gaa atg gag cag ttt 1613 Val Tyr Asp Phe Val Gly Leu Leu Val
Ser Pro Glu Met Glu Gln Phe 395 400 405 gcc ctg aac atg acc agt ggc
tcg ggc tcc tcc tcc aag cct ttc gtg 1661 Ala Leu Asn Met Thr Ser
Gly Ser Gly Ser Ser Ser Lys Pro Phe Val 410 415 420 gta tct gca cgc
aat gcc acg cag gcc gcc aat gcc tgg cgg acc gcc 1709 Val Ser Ala
Arg Asn Ala Thr Gln Ala Ala Asn Ala Trp Arg Thr Ala 425 430 435 440
ctc acc ttc cag cag atc aaa cag gtg gag gag ttt tgc tac cag ccc
1757 Leu Thr Phe Gln Gln Ile Lys Gln Val Glu Glu Phe Cys Tyr Gln
Pro 445 450 455 atg gcc gtc ctg ggc tat gag cgg gtc aac agc cct gag
gag gtc aaa 1805 Met Ala Val Leu Gly Tyr Glu Arg Val Asn Ser Pro
Glu Glu Val Lys 460 465 470 gac ctc agc aag acc ctg ctt cgg aag ccc
cgt ctc taaaaggggt 1851 Asp Leu Ser Lys Thr Leu Leu Arg Lys Pro Arg
Leu 475 480 tcccaggaga cctgattccc tgtggtgata cctataaaga ggatcgtagt
gtgtttaaat 1911 aaacacagtc cagactcaaa cggaggaagc ccacatattc
tattatagat atataaataa 1971 tcacacacac acttgctgtc aatgttttga
gtcagtgcat ttcaaggaac agccacaaaa 2031 tacacacccc taagaaaagg
caagacttga acgttctgac caggtgcccc tcttcttctt 2091 tgccttctct
tgtcctcttt ctcctatttc ttaccctgtc ctccacctgc cttccatttt 2151
gaagtgggat gttaatgaaa tcaagttcca gtaacccaaa tcttgtttac aaaatattcg
2211 tggtatctgt gaacatgtta agagtaattt ggatgtgggg gtgggggtgg
agaaagggga 2271 agtggtccag aaacaaaaag ccccattggg catgataagc
cgaggaggca ttcttcctaa 2331 aagtagactt ttgtgtaaaa agcaaaggtt
acatgtgagt attaataaag aagataataa 2391 ataaaaaaaa aaaaaaaa 2409 4
484 PRT Homo sapiens 4 Met Lys Val Phe Arg Arg Lys Ala Leu Val Leu
Cys Ala Gly Tyr Ala 1 5 10 15 Leu Leu Leu Val Leu Thr Met Leu Asn
Leu Leu Asp Tyr Lys Trp His 20 25 30 Lys Glu Pro Leu Gln Gln Cys
Asn Pro Asp Gly Pro Leu Gly Ala Ala 35 40 45 Ala Gly Ala Ala Gly
Gly Lys Leu Gly Ala Pro Arg Ala Ala Ser Gly 50 55 60 Arg Ala Ala
Pro Cys Ser Cys Pro Phe Gly Pro Pro His Ser Leu Pro 65 70 75 80 Pro
Ser Arg Cys Arg Arg Arg Gly Asp Thr Leu Gln Pro Arg Gln Gly
85 90 95 Trp Arg Gly Leu Arg Pro Leu Gln Ala Met Ala Leu Gly Ala
Pro Glu 100 105 110 Gly Val Gly Asp Lys Arg His Trp Met Tyr Val Phe
Thr Thr Trp Arg 115 120 125 Ser Gly Ser Ser Phe Phe Gly Glu Leu Phe
Asn Gln Asn Pro Glu Val 130 135 140 Phe Phe Leu Tyr Glu Pro Val Trp
His Val Trp Gln Lys Leu Tyr Pro 145 150 155 160 Gly Asp Ala Val Ser
Leu Gln Gly Ala Ala Arg Asp Met Leu Ser Ala 165 170 175 Leu Tyr Arg
Cys Asp Leu Ser Val Phe Gln Leu Tyr Ser Pro Ala Gly 180 185 190 Ser
Gly Gly Arg Asn Leu Thr Thr Leu Gly Ile Phe Gly Ala Ala Thr 195 200
205 Asn Lys Val Val Cys Ser Ser Pro Leu Cys Pro Ala Tyr Arg Lys Glu
210 215 220 Val Val Gly Leu Val Asp Asp Arg Val Cys Lys Lys Cys Pro
Pro Gln 225 230 235 240 Arg Leu Ala Arg Phe Glu Glu Glu Cys Arg Lys
Tyr Arg Thr Leu Val 245 250 255 Ile Lys Gly Val Arg Val Phe Asp Val
Ala Val Leu Ala Pro Leu Leu 260 265 270 Arg Asp Pro Ala Leu Asp Leu
Lys Val Ile His Leu Val Arg Asp Pro 275 280 285 Arg Ala Val Ala Ser
Ser Arg Ile Arg Ser Arg His Gly Leu Ile Arg 290 295 300 Glu Ser Leu
Gln Val Val Arg Ser Arg Asp Pro Arg Ala His Arg Met 305 310 315 320
Pro Phe Leu Glu Ala Ala Gly His Lys Leu Gly Ala Lys Lys Glu Gly 325
330 335 Val Gly Gly Pro Ala Asp Tyr His Ala Leu Gly Ala Met Glu Val
Ile 340 345 350 Cys Asn Ser Met Ala Lys Thr Leu Gln Thr Ala Leu Gln
Pro Pro Asp 355 360 365 Trp Leu Gln Gly His Tyr Leu Val Val Arg Tyr
Glu Asp Leu Val Gly 370 375 380 Asp Pro Val Lys Thr Leu Arg Arg Val
Tyr Asp Phe Val Gly Leu Leu 385 390 395 400 Val Ser Pro Glu Met Glu
Gln Phe Ala Leu Asn Met Thr Ser Gly Ser 405 410 415 Gly Ser Ser Ser
Lys Pro Phe Val Val Ser Ala Arg Asn Ala Thr Gln 420 425 430 Ala Ala
Asn Ala Trp Arg Thr Ala Leu Thr Phe Gln Gln Ile Lys Gln 435 440 445
Val Glu Glu Phe Cys Tyr Gln Pro Met Ala Val Leu Gly Tyr Glu Arg 450
455 460 Val Asn Ser Pro Glu Glu Val Lys Asp Leu Ser Lys Thr Leu Leu
Arg 465 470 475 480 Lys Pro Arg Leu 5 20 DNA Artificial Sequence
Primer 5 gtcgtcggac tggtggacga 20 6 20 DNA Artificial Sequence
primer 6 cccagagcgt ggtagtctgc 20 7 29 DNA Artificial Sequence
primer 7 acgaattcgg gatgaaggta tttcgcagg 29 8 29 DNA Artificial
Sequence primer 8 atgaattctc aaagccgggg cttcctgag 29 9 27 DNA
Artificial Sequence primer 9 ctgaattcgg aatgaaggtg ttccgta 27 10 27
DNA Artificial Sequence primer 10 gagaattctt agagacgggg cttccga
27
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