U.S. patent application number 09/117860 was filed with the patent office on 2001-09-27 for novel beta1-4 n-acetylglucosaminyltransferase and gene encoding.
Invention is credited to MINOWA, MARI, OGURI, SUGURU, TAKEUCHI, MAKOTO, TANIGUCHI, NAOYUKI, YOSHIDA, ARUTO.
Application Number | 20010024814 09/117860 |
Document ID | / |
Family ID | 26487595 |
Filed Date | 2001-09-27 |
United States Patent
Application |
20010024814 |
Kind Code |
A1 |
OGURI, SUGURU ; et
al. |
September 27, 2001 |
NOVEL BETA1-4 N-ACETYLGLUCOSAMINYLTRANSFERASE AND GENE ENCODING
Abstract
The present invention relates to a novel enzyme having
.beta.1.fwdarw.4 N-acetylglucosaminyltransferase (GnT-IV) activity;
a gene encoding the enzyme; a recombinant DNA comprising the gene;
a host cell comprising the recombinant DNA; a method for producing
an enzyme protein having GnT-IV activity comprising culturing the
host cell in a medium; and a saccharide in which the sugar chain is
modified using a GnT-IV. According to the present invention, a
novel GnT-IV, a method for producing the enzyme and a gene coding
for the enzyme are provided. With the GnT-IV of the present
invention, it has become possible to produce a saccharide having a
branching structure which could not be formed with conventional
glycosyltransferases. Thus, the GnT-IV of the invention is useful
not only for producing or improving glycoconjugate type
pharmaceuticals, reagents and foods, but also for modifying the
sugar chain structure of any biopolymer.
Inventors: |
OGURI, SUGURU; (HOKKAIDO,
JP) ; MINOWA, MARI; (KANAGAWA, JP) ; YOSHIDA,
ARUTO; (KANAGAWA, JP) ; TANIGUCHI, NAOYUKI;
(OSAKA, JP) ; TAKEUCHI, MAKOTO; (KANAGAWA,
JP) |
Correspondence
Address: |
FOLEY & LARDNER
WASHINGTON HARBOUR
3000 K STREET NW SUITE 500
WASHINGTON
DC
200075109
|
Family ID: |
26487595 |
Appl. No.: |
09/117860 |
Filed: |
August 12, 1998 |
PCT Filed: |
December 10, 1997 |
PCT NO: |
PCT/JP97/04546 |
Current U.S.
Class: |
435/193 ;
435/252.3; 435/320.1; 435/325; 435/97; 536/22.1; 536/23.2 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 9/1051 20130101 |
Class at
Publication: |
435/193 ;
435/320.1; 435/325; 435/252.3; 435/97; 536/22.1; 536/23.2 |
International
Class: |
C12N 009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 12, 1996 |
JP |
8/332411 |
Jun 18, 1997 |
JP |
9/161462 |
Claims
1. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase having an
activity to produce a saccharide having a partial structure
represented by the formula below: 23using UDP-GlcNAc as a sugar
donor and a saccharide having a partial structure represented by
the formula below as a sugar receptor: 24
2. The .beta.1.fwdarw.4 N-acetylglucosaminyltransferase of claim 1,
wherein the saccharide as a sugar receptor is an oligosaccharide,
polysaccharide, glycoconjugate (glycopeptide, glycoprotein,
glycolipid or proteoglycan) or a derivative thereof.
3. The .beta.1.fwdarw.4 N-acetylglucosaminyltransferase of claim 1,
wherein the sugar receptor is a saccharide having the partial
structure represented by the formula below: 25wherein R1: H--,
Man.alpha.1.fwdarw.6 or GlcNAc.beta.1.fwdarw.6 R2: H--,
Man.alpha.1.fwdarw.3 or GlcNAc.beta.1.fwdarw.2 R3: --OH or
.beta.1.fwdarw.4GlcNAc.fwdarw.R4 R4: --OH, --H, -pyridylamine or
-peptide chain.
4. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase comprising
the amino acid sequences shown in SEQ ID NOS: 1-14 as partial
sequences.
5. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase consisting of
the amino acid sequence shown in SEQ ID NO: 18 or the amino acid
sequence shown in SEQ ID NO: 18 which has addition, deletion or
substitution of one or more amino acid residues and yet which
produces .beta.1.fwdarw.4 N-acetylglucosaminyltransferase
activity.
6. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase consisting of
a partial sequence of the amino acid sequence shown in SEQ ID NO:
18 including at least from position 93 to position 383 residues, or
said partial sequence which has addition, deletion or substitution
of one or more amino acid residues and yet which produces
.beta.1.fwdarw.4 N-acetylglucosaminyltransferase activity.
7. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase consisting of
the amino acid sequence shown in SEQ ID NO: 24 or the amino acid
sequence shown in SEQ ID NO: 24 which has addition, deletion or
substitution of one or more amino acid residues and yet which
produces .beta.1.fwdarw.4 N-acetylglucosaminyltransferase
activity.
8. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase consisting of
a partial sequence of the amino acid sequence shown in SEQ ID NO:
24 including at least from position 94 to position 383 residues, or
said partial sequence which has addition, deletion or substitution
of one or more amino acid residues and yet which produces
.beta.1.fwdarw.4 N-acetylglucosaminyltransferase activity.
9. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase consisting of
the amino acid sequence shown in SEQ ID NO: 37 or the amino acid
sequence shown in SEQ ID NO: 37 which has addition, deletion or
substitution of one or more amino acid residues and yet which
produces .beta.1.fwdarw.4 N-acetylglucosaminyltransferase
activity.
10. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase consisting
of a partial sequence of the amino acid sequence shown in SEQ ID
NO: 37 including at least from position 91 to position 390
residues, or said partial sequence which has addition, deletion or
substitution of one or more amino acid residues and yet which
produces .beta.1.fwdarw.4 N-acetylglucosaminyltransferase
activity.
11. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase gene coding
for the .beta.1.fwdarw.4 N-acetylglucosaminyltransferase of claim 5
or 6.
12. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase gene coding
for the .beta.1.fwdarw.4 N-acetylglucosaminyltransferase of claim 7
or 8.
13. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase gene coding
for the .beta.1.fwdarw.4 N-acetylglucosaminyltransferase of claim 9
or 10.
14. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase gene
consisting of the nucleotide sequence shown in SEQ ID NO: 17.
15. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase gene
consisting of the nucleotide sequence shown in SEQ ID NO: 23.
16. A .beta.1.fwdarw.4 N-acetylglucosaminyltransferase gene
consisting of the nucleotide sequence shown in SEQ ID NO: 36.
17. A recombinant DNA obtained by inserting into a vector DNA the
.beta.1.fwdarw.4 N-acetylglucosaminyltransferase gene of any one of
claims 11 to 16.
18. A chromosomal fragment comprising a part or all of the
.beta.1.fwdarw.4 N-acetylglucosaminyltransferase gene of any one of
claims 11 to 16.
19. A host cell carrying the recombinant DNA of claim 17.
20. A host cell into which the chromosomal fragment of claim 18 is
artificially introduced.
21. A method for purifying the .beta.1.fwdarw.4
N-acetylglucosaminyltransf- erase of any one of claims 1 to 3 from
biological samples.
22. A method for producing a .beta.1.fwdarw.4
N-acetylglucosaminyltransfer- ase comprising culturing the host
cell of claim 19 in a medium and recovering the .beta.1.fwdarw.4
N-acetylglucosaminyltransferase from the resultant culture.
23. A method for producing a .beta.1.fwdarw.4
N-acetylglucosaminyltransfer- ase comprising culturing the host
cell of claim 20 in a medium and recovering the .beta.1.fwdarw.4
N-acetylglucosaminyltransferase from the resultant culture.
24. A method for producing a .beta.1.fwdarw.4
N-acetylglucosaminyltransfer- ase comprising recovering the
.beta.1.fwdarw.4 N-acetylglucosaminyltransfe- rase from the
secreta, body fluid or homogenate originated from the host cell of
claim 19.
25. A method for producing a .beta.1.fwdarw.4
N-acetylglucosaminyltransfer- ase comprising recovering the
.beta.1.fwdarw.4 N-acetylglucosaminyltransfe- rase from the
secreta, body fluid or homogenate originated from the host cell of
claim 20.
26. A saccharide of which the sugar chain structure is modified
with the .beta.1.fwdarw.4 N-acetylglucosaminyltransferase of any
one of claims 1 to 3.
27. The saccharide of claim 26, which is an oligosaccharide, a
glycopeptide, a glycoprotein or a derivative thereof.
28. A method for modifying a branching structure of a sugar chain
of a glycoprotein produced by a host cell, comprising introducing
into the host cell the .beta.1.fwdarw.4
N-acetylglucosaminyltransferase gene of any one of claims 11 to
16.
29. A glycoprotein in which a branching structure of a sugar chain
is modified by the method of claim 28.
30. The glycoprotein of claim 29, which is erythropoietin.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel
N-acetylglucosaminyltransf- erase (GlcNAc transferase) which
recognizes a specific sugar chain structure in a saccharide and
introduces thereinto a GlcNAc .beta.1.fwdarw.4 branching
structure.
BACKGROUND ART
[0002] 1. Glycoproteins
[0003] Most of proteins occurring in nature are not simple proteins
composed of amino acids alone, but "mature" proteins having sugar
chains and other substances such as phosphates and lipids attaching
thereto. Therefore, the development of simple protein-type products
produced by Escherichia coli as a host has involved various
problems because such products lack the maturing process of
proteins. Since all of secretion-type physiologically active
proteins (e.g. cytokines) are glycoproteins with a few exceptions,
the function and the role of sugar chains have attracted attention
as the most important point in the development of biological
pharmaceuticals.
[0004] Sugar chains in glycoproteins are classified roughly into
Asn-linked type, mucin-type, O-linked GlcNAc type, GPI anchor type
and proteoglycan type [Makoto Takeuchi, "Glycobiology Series 5:
Glycotechnology", Kihata, Hakomori and Nagai (eds.), Kodansha
Scientific Co., (1994), 191-208]. Each of these types of sugar
chains has its own biosynthesis pathway and a discrete
physiological function. Asn-linked sugar chains are distributed
widely in molds, yeasts, insects, plants and animals. The basic
biosynthesis pathway for Asn-linked sugar chains is conserved
beyond species (FIG. 1). A sugar chain(s) characteristic of a
specific species is(are) formed on the outer side (called the
"non-reducing terminal side") of the core sugar chain moiety which
is common in the biosynthesis of Asn-linked sugar chains. A
mannan-type sugar chain in which .alpha.1,3- and
.alpha.1,2-branching mannose residues attach to a main chain
extending via .alpha.1,6 linkages is a sugar chain structure
characteristic of fungi such as yeasts (see Panel a in FIG. 2)
[Hiroshi Nakajima, Sugar Chain Technology, Industry Survey
Association (1992), 384-397]. On the other hand, in insects, plants
and animals, extension of mannose residues is not observed;
instead, a high mannose type sugar chain is formed which is a sugar
chain transferred from a dolichol intermediate and only trimmed
(see Panel c in FIG. 2). A unique structure having characteristic
xylose or the like (see Panel b in FIG. 2) is also observed in
insects, plants and mollusks. In animals, characteristic sugar
chain structures such as complex type sugar chain (Panel e in FIG.
2) and hybrid type sugar chain (Panel d in FIG. 2) are observed; in
the former, GlcNAc branching structures are formed in a once
trimmed sugar chain, and addition of other kinds of monosaccharides
such as galactose and sialic acid forms complicated structures; in
the latter, both a complex type sugar chain and a high mannose type
sugar chain are present [Kiyoshi Furukawa, Sugar Chain Technology,
Industry Survey Association (1992), 64-75].
[0005] Such sugar chains as described above are conferred on most
of cell surface proteins and secretion proteins, and are thought to
play important roles which determine the natures and properties of
cells and proteins. Among all, the portion of a sugar chain
structure which forms a branch elongating like antennas from the
common core sugar chain is called a sugar chain branching
structure. This structure is believed to have a function to give an
organism recognition ligand (i.e., the end portion of the sugar
chain) a high degree of freedom to thereby provide chances for
multipoint recognition and another function to maximize the
protection ability for the protein moiety by greatly increasing the
space-occupying volume (Takeuchi et al., supra). Therefore, by
controlling the branching structure of sugar chains, it is possible
to modify the physiological functions, sush as in vivo stability,
in vivo kinetics and organ-targeting properties of glycoproteins in
various ways. In view of this, technology to control branching
structures of sugar chains is expected as biotechnology of the next
generation for the development of glycoprotein-type pharmaceuticals
which are "tender to humans".
[0006] 2. Physiological Significance of Glycoprotein Sugar
Chains
[0007] Sugar chains of secretion type glycoproteins exhibit
excellent functions in biosynthesis, intracellular sorting, masking
of antigenicity, in vivo stability and organ-targeting properties
of glycoproteins. Sugar chains of cell surface proteins are known
to change in response to changes in cells (such as differentiation,
change to a morbid state, canceration). In particular, it has been
reported that there is a close relation between the metastasis of
cancer and the branching structure of sugar chains.
[0008] (1) Masking of Antigenicity
[0009] It is considered that sugar chains have a high degree of
freedom in terms of steric structure and thus are moving freely
like propellers. Therefore, protein molecules such as proteases and
antibodies against proteins not having affinity to sugar chains are
shook off by the sugar chains and thus cannot gain access to the
protein moiety. As a result, even if there is antigenicity in the
peptide moiety near the sugar chain binding site, antibody
molecules cannot have access to the peptide moiety. Thus, an
antigen-antibody reaction is extremely difficult to occur. Further,
when a glycoprotein has been captured by a macrophage and the
degradation products are presented as antigen, receptors are
difficult of access to the peptides around the sugar chain binding
site. Thus, antigenic stimulation is difficult to occur. Actually,
it is reported that when sugar chains have been introduced into the
central portion of the antigenic peptide of ovalbumin lysozyme, the
binding of MHC class II molecules to the antigen is remarkably
inhibited [Mouritsen, S., Meldal, M., Christiansen-Brams, I.,
Elsner, H. and Werdelin, O., Eur. J. Immunol., (1994), 24,
1066-10721. The effect of such masking of antigenicity becomes
greater as the volume occupied by sugar chains is greater. Thus, it
is considered that the development of a branching structure
contributes to the effect of such masking greatly.
[0010] (2) In Vivo Stability
[0011] With respect to erythropoietin which is the first
glycoprotein-type pharmaceutical ever produced from a transgenic
animal cell as a host, the functions of sugar chains thereof have
been studied thoroughly. As a result, it has been shown that the
sugar chains of erythropoietin work inhibitorily against the
binding of erythropoietin with its receptor but make a decisive
contribution to the retaining of the active structure and the
improvement of in vivo kinetics; as a whole, the sugar chains have
been shown to be essential for expression of the pharmacological
activity of erythropoietin (Takeuchi, M. and Kobata, A.,
Glycobiology (1991), 1, 337-346]. In particular, a strong
correlation between the number of antennae in sugar chains and the
pharmacological effect of erythropoietin has been found, and thus
the importance of its branching structure (a branching structure
formed by GlcNAc residues attaching to the core sugar chain) which
never attracted attention has been made clear for the first time
[Takeuchi, M., Inoue, N., Strickland, T. W., Kobata, M., Wada, M.,
Shimizu, R., Hoshi, S., Kozutsumi, H., Takasaki, S. and Kobata, A.,
Proc. Natl. Acad. Sci. USA, (1989), 86, 7819-22]. The major cause
of the above phenomenon is explained as follows: erythropoietin
without developed branching structure is cleared rather rapidly in
kidney and, as a result, the in vivo residence time of such
erythropoietin becomes shorter [Misaizu, T., Matsuki, S.,
Strickland, T. W., Takeuchi, M., Kobata, A. and Takasaki, S.,
Blood, (1995), 86, 4097-4104].
[0012] (3) Organ Targeting Property
[0013] Most of biological tissues have lectin-like receptors and
use then in cell-cell interactions or to uptake glycoproteins from
blood. The asialoprotein-binding lectin in liver is a
representative example of a clearance system for aged glycoproteins
[Toshihiro Kawasaki, Sugar Chain Technology, Industry Survey
Association (1992), 125-136]. In addition, selectin contained in
vascular endothelial cells, platelets and leucocytes (Kawasaki,
supra) and the lectin receptor present on the surface of
macrophages and NK cells (Kawasaki, supra) are well known.
Furthermore, not only glycoproteins but also cells are known to
gather in a specific tissue using sugar chains as ligands. Cases of
the homing of bone marrow cells [Tatsuo Irimura, "Glycobiology
Series 3: Glycobiology in Cell Society", Katsutaka Nagai, Senichiro
Hakomori and Akira Kobata (eds.), Kodansha Scientific Co., (1993),
127-175] and the recruiting of neutrophiles to inflammatory sites
(Irimura, supra) are examined in detail. Putting all these things
together, it can be well assumed that glycoproteins and cells have,
via their sugar chain structures, a targeting property toward
specific organs or tissues presenting a lectin receptor in blood
circulation, although such a targeting system is not found in all
organ. This means that drug delivery by means of sugar chains is
possible. In such drug delivery, the affinity of lectin for sugar
chains is greatly influenced by the degree of freedom and the
number of sugar chain ligands. Therefore, the branching structure
of sugar chains will be the most important point in such drug
delivery.
[0014] (4) Correlation between Cells' Change into Morbid State and
Sugar Chain Branched Structure thereof [Junko Kato, Naoko Suzuki,
"Sugar Chain Technology and Development of Pharmaceutical",
Foundation for the Relief and Study of Injury Caused by
Pharmaceutical Side Effect (ed.), Yakugyo-Jiho-Sha, (1994),
107-13214]
[0015] Once a plant lectin called L-PHA was developed as a probe to
detect a multi-branching type sugar chain structure, it has become
possible to examine various morbid tissue samples. As a result, a
tendency has been found that some types of cancer cells,
particularly, cancer cells with a high metastasis ability are
stained well with L-PHA. Thus, researchers have become aware of the
correlation between the branching structure of sugar chains and the
metastasis ability of cancer cells. Human chorionic gonadotropin
(hCG) is a glycoprotein hormone vigorously biosynthesized in villus
tissues at an early stage of pregnancy. Since a considerable amount
of hCG is discharged into urine, hCG is clinically utilized as an
indicator of pregnancy. The Asn-linked sugar chains mainly formed
by the mono- and biantennary complex type chains are characteristic
to hCG. As cancer increases its malignancy from trophoblastoma to
invasive mole and from invasive mole to choriocarcinoma, it is
reported that 2,4,2 type tri-antennary sugar chains and abnormal
biantennary sugar chains (both are formed through the action of
GnT-IV on normal biantennary and mono-antennary sugar chains,
respectively) appear in the sugar chains of hCG [Katsuko Yamashita,
Protein, Nucleic Acid and Enzyme (1992), 37, 1880-1888]. As a cause
of this phenomenon, it is suggested that the activity of GnT-IV
increases as the malignancy of choriocarcinoma progresses.
[0016] .gamma.-Glutamyltranspeptidase (.gamma.-GTP) is a
glycoprotein occurring specifically abundant in liver. Since the
serum .gamma.-GTP level drastically increases when there is a liver
disease, this level is used as clinical indicator of a liver
disease. Further, Yamashita et al. [Yamashita, K., Totani, K.,
Iwaki, Y., Takamisawa, I., Takeishi, N., Higashi, T., Sakamoto, Y.
and Kobata, A., J. Biochem., (1989), 105, 728-735] have found that,
as a result of canceration of cells, the sugar chain structure of
.gamma.-GTP changes abnormally in its branching structure similar
to those in abnormal hCG; thus, they have reported the correlation
between canceration and the activation of GnT-IV. The Asn-linked
sugar chains of .gamma.-GTP derived from healthy human liver cells
are composed mainly of the biantennary complex type sugar chain
with small amounts of tri-antennary and tetra-antennary sugar
chains mixed therein. In contrast, a remarkable increase in the
degree of branching was observed in the Asn-linked sugar chains of
.gamma.-GTP derived from human hepatoma cells. At the same time,
though small in amounts, high mannose type sugar chains and
abnormal biantennary sugar chains (both of which were not observed
in .gamma.-GTP from normal cells) appeared. As a cause of these
changes in sugar chain structure, a possibility is suggested that
N-acetylglucosaminyltransferase IV (GnT-IV) and V (GnT-V) are
activated in relation to canceration of liver cells (Yamashita et
al., supra).
[0017] It is also reported that the sugar chain branching structure
of a glycoprotein in cells is greatly changed by viral infection
(Yamashita et al., supra). BHK cells have sugar chain structures
with branching up to tetraantennary type. When BHK cells are
transformed with polyomavirus, biantennary type sugar chains
decrease in the glycoprotein sugar chains produced by the cells,
while tetraantennary type sugar chains and the N-acetyllactosamine
repeat structures increase; as a whole, a remarkable increase in
the number of branches was recognized [Takasaki S., Ikehira, H. and
Kobata A., Biochem. Biophys. Res. Commun., (1980), 90, (3),
735-742]. As a cause of the above change, activation of GnT-IV,
GnT-V and i-GnT may be considered.
[0018] 3. Enzymes relating to the Sugar Chain Branching Structures
of Glycoproteins
[0019] The complex type sugar chain which is a glycoprotein sugar
chain structure characteristic of animals has a complicated
branching structure in which N-acetylglucosamine (GlcNAc) residues
are attaching to the common core structure in various manners
(Kiyoshi Furukawa, supra) (FIG. 1). Since this branching structure
is closely related to in vivo and in vivo stability, localization,
biological activity and pharmacological property of glycoproteins
(Makoto Takeuchi, supra), the process of biosynthesis of the
branching structure has been investigated in detail. By using
inventive substrates H. Schachter et al. have discriminated the
various enzyme activities in hen oviduct to thereby predict the
presence of GlcNAc branch forming enzymes from GnT-I to GnT-VI
(group of GlcNAc glycosyltransferases; FIG. 3) [Glesson, P. A. and
Schachter, H., J. Biol. Chem., (1983), 258, 6162-6173]. Thereafter,
GnT-I [Kumar, R., Yang, J., Larsen, R. D. and Stanley P., Proc.
Natl. Acad. Sci. USA, (1990), 87, 9948-9952; Sarkar, M., Hull, E.,
Nishikawa, Y., Simpson, R. J., Moritz, R. L., Dunn, R. and
Schachter, H., Proc. Natl. Acad. Sci., USA, (1991), 88, 234-238],
GnT-II [D'Agostaro, G A., Zingoni, A., Moritz, R L., Simpson, R J.,
Schachter, H. and Bendiak, B., J. Biol. Chem., (1995), 270,
15211-21], GnT-III [Nishikawa, A., Ihara, Y., Hatakeyama, M.,
Kangawa, K. and Taniguchi, N., J. Biol. Chem., (1992), 267,
18199-18204] and GnT-V [Shorebah, M. G., Hindsgaul, O. and Pierce,
M., J. Biol. Chem., (1992), 267, 2920-2927; Gu, J., Nishikawa, A.,
Turuoka, N., Ono, M., Yamaguchi, N., Kangawa, K. and Taniguchi, N.,
J. Biochem., (1993), 113, 614-619] were successively purified, and
the genes thereof were cloned. However, with these known GlcNAc
transferases alone, it is impossible to form the main sugar chain
(tetraantennary type; see the formula below) found in .alpha.1 acid
glycoprotein known as a representative human blood glycoprotein
[Yoshima, K., Tsuji, T., Irimura, T. and Osawa, T, J. Biol. Chem.,
(1984), 256, 10834-10840] and erythropoietin [Takeuchi, M.,
Takasaki, S., Shimada, M. and Kobata, A., J. Biol. Chem., (1990),
265, 12127-12130]. Therefore, an N-acetylglucosaminyltransferase
having such substrate specificity and reaction specificity that are
expected in GnT-IV has been searched for as a missing link. 1
[0020] In addition to those mentioned above, the following
N-acetylglucosaminyltransferases have been purified or the genes
thereof have been cloned: a transferase acting on mucin-type sugar
chains (Bierhuizen, M. F., Maemura, K. and Fukuda, M., J. Biol.
Chem., (1994), 269, 4473-4479], a transferase acting on
glycolipids, and a transferase forming the sugar chain epitope
known as I.multidot.i antigenic structure [Kawashima, H., Yamamoto,
K., Osawa, T. and Irimura, T., J. Biol. Chem., (1993), 268,
27118-27126; Bierhuizen, M. F., Mattei, M. G. and Fukuda, M., Genes
Dev., (1993), 7, 468-478]. However, the substrate specificity of
these transferases and the mode of binding of the GlcNAc group
transferred by these transferases are different from those of
GnT-IV. Any of these transferases does not yield products
resembling GnT-IV products.
DISCLOSURE OF THE INVENTION
[0021] It is an object of the present invention to provide an
enzyme having .beta.1.fwdarw.4 N-acetylglucosaminyltransferase
(hereinafter referred to as "GnT-IV") activity; a gene encoding the
enzyme; a recombinant DNA comprising the gene; a cell containing
the recombinant DNA; a method for producing an enzyme protein
having GnT-IV activity comprising culturing the cell in a medium;
and a saccharide in which the sugar chains are modified with
GnT-IV.
[0022] Toward the solution of the above assignments, the present
inventors have made intensive and extensive researches. As a
result, the inventors have isolated and purified a GnT-IV enzyme
protein from bovine small intestine, and characterized the
biochemical properties of the protein. Then, the inventors have
succeeded in cloning a gene coding for bovine GnT-IVa from a cDNA
library and mRNA from the small intestine based on a partial amino
acid sequence of the above enzyme protein. Further, based on bovine
GnT-IVa gene, the inventors have succeeded in cloning two genes
coding for human GnT-IVa and human GnT-IVb, from cDNA libraries and
mRNAs from human liver and human lung, respectively. The present
invention has been completed by confirming that the products of
these genes exhibit GnT-IV activity.
[0023] The first invention of the present application relates to a
GnT-IV having an activity to produce a saccharide having a partial
structure represented by the formula below: 2
[0024] using UDP-GlcNAc as a sugar donor and a saccharide having a
partial structure represented by the formula below as a sugar
receptor: 3
[0025] The second invention relates to a GnT-IV consisting of the
amino acid sequence shown in SEQ ID NO: 18 or the amino acid
sequence shown in SEQ ID NO: 18 which has addition, deletion or
substitution of one or more amino acid residues and yet which
produces GnT-IV activity; a GnT-IV consisting of the amino acid
sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in
SEQ ID NO: 24 which has addition, deletion or substitution of one
or more amino acid residues and yet which produces GnT-IV activity;
and a GnT-IV consisting of the amino acid sequence shown in SEQ ID
NO: 37 or the amino acid sequence shown in SEQ ID NO: 37 which has
addition, deletion or substitution of one or more amino acid
residues and yet which produces GnT-IV activity.
[0026] The third invention relates to a GnT-IV gene coding for a
GnT-IV consisting of the amino acid sequence shown in SEQ ID NO: 18
or the amino acid sequence shown in SEQ ID NO: 18 which has
addition, deletion or substitution of one or more amino acid
residues and yet which produces GnT-IV activity; a GnT-IV gene
coding for a GnT-IV consisting of the amino acid sequence shown in
SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24
which has addition, deletion or substitution of one or more amino
acid residues and yet which produces GnT-IV activity; a GnT-IV gene
coding for a GnT-IV consisting of the amino acid sequence shown in
SEQ ID NO: 37 or the amino acid sequence shown in SEQ ID NO: 37
which has addition, deletion or substitution of one or more amino
acid residues and yet which produces GnT-IV activity; a GnT-IV gene
consisting of the nucleotide sequence shown in SEQ ID NO: 17; a
GnT-IV gene consisting of the nucleotide sequence shown in SEQ ID
NO: 23; and a GnT-IV gene consisting of the nucleotide sequence
shown in SEQ ID NO: 36.
[0027] The fourth invention relates to a recombinant DNA obtainable
by inserting any of the above GnT-IV gene into a vector DNA; and a
chromosomal fragment comprising a part or all of any one of the
above GnT-IV gene.
[0028] The fifth invention relates to a host cell carrying the
above recombinant DNA; and a host cell into which the above
chromosomal fragment is artificially introduced.
[0029] The sixth invention relates to a method for producing a
GnT-IV comprising culturing the above host cell in a medium and
recovering the GnT-IV from the resultant culture; and a method for
producing a GnT-IV comprising recovering the GnT-IV enzyme from the
secreta, body fluids or homogenete originated from the above host
cell.
[0030] The seventh invention relates to a method for purifying the
GnT-IV from biological samples.
[0031] The eighth invention relates to a saccharide of which the
sugar chain structure is modified with the GnT-IV.
[0032] Hereinbelow, the present invention will be described in
detail.
[0033] The GnT-IV gene of the invention can be isolated as
described below.
[0034] Isolation of Bovine GnT-IVa Gene
[0035] First, a microsome fraction from bovine small intestine
solubilized with a detergent is subjected to a series of
purification procedures using anion exchange chromatography, copper
chelate chromatography, two-step affinity chromatography using a
substrate analogue and gel filtration to thereby obtain a purified
sample of GnT-IV enzyme. The resultant purified sample is subjected
to SDS-PAGE and then transferred onto a PVDF membrane. The
transferred protein, as it is or after restricted hydrolysis, is
analyzed with a gas phase amino acid sequencer to obtain a partial
amino acid sequence for the GnT-IV enzyme.
[0036] Subsequently, an RT-PCR is performed on the RNA extracted
from the animal cells (i.e., bovine small intestine) as a template
using primers designed based on the partial amino acid sequences
determined above. Further, using a fragment obtained by the RT-PCR
as a probe, the GnT-IV gene of interest is screened from a cDNA
library from the above-mentioned tissue by plaque hybridization. A
cDNA fragment contained in the resultant positive plaque is cut out
and subcloned into a vector such as pUC19, followed by analysis of
the nucleotide sequence thereof. If the full length of the gene
coding for the protein of interest is not contained in the
fragment, plaque hybridization is performed again using a part of
the subcloned cDNA fragment as a probe. Alternatively, terminal
portions of the cDNA of interest are obtained by RACE or the like
based on the information on the nucleotide sequence obtained above.
The thus obtained GnT-IV gene (which is named GnT-IVa aferward) is
subjected to analysis of its entire nucleotide sequence.
Subsequently, the amino acid sequence is translated from the gene
having the above-mentioned nucleotide sequence. This amino acid
sequence is as shown in SEQ ID NO: 18.
[0037] Isolation of Human GnT-IVa and GnT-IVb Genes
[0038] Human GnT-IVa and GnT-IVb genes can be obtained by
performing a RT-PCR using RNA extracted from a human tissue (liver
or lung) and based on the information on the nucleotide sequence of
bovine GnT-IVa gene as obtained above, followed by screening of a
cDNA library from the above tissue. The resultant human GnT-IVa and
GnT-IVb genes are subjected to analysis of their entire nucleotide
sequences. Subsequently, the amino acid sequences are translated by
these genes.
[0039] These amino acid sequences are as shown in SEQ ID NOS: 24
and 37.
[0040] In order to obtain a DNA coding for the amino acid sequence
shown in SEQ ID NO: 18, 24 or 37 having addition, deletion or
substitution of one or more amino acid residues, a number of
methods may be used. For example, a method of treating DNA with a
mutagen to induce point mutation or a deletion mutation; a method
comprising cleaving DNA selectively, removing or adding a selected
nucleotide and then ligating DNA; site-specific mutagenesis; and
the like may be enumerated.
[0041] The GnT-IV protein of the invention can be produced by
preparing a recombinant vector into which a DNA coding for the
GnT-IV of the invention obtained by the method described above is
inserted downstream of a promoter, introducing the vector into a
host cell and culturing the resultant cell. The vector DNA used for
this purpose may be either plasmid DNA or bacteriophage DNA. For
example, pSVL vector (Pharmacia, Sweden) shown in an Example
described later may be used. As the host cell into which the
resultant recombinant DNA is introduced, any cell that is
conventionally used in recombinant DNA techniques may be used, for
example, a prokaryotic cell, an animal cell, a yeart, a fungi, an
insect cell. Specific examples include Escherichia coli as a
prokaryotic cell and CHO cells from chinese hamster or COS cells
from monkey as an animal cell.
[0042] The transformation of the host cell described above is
performed by conventional methods for each host. For example, if
the host is E. coli, a vector comprising the recombinant DNA is
introduced by the heat shock method or electroporation into
competent cells prepared by the calcium method or the like. If the
host is yeast, a vector comprising the recombinant DNA is
introduced by the heat shock method or electroporation into
competent cells prepared by the lithium method or the like. If the
host is an animal cell, a vector comprising the recombinant DNA is
introduced into the cell at the growth phase or the like by the
calcium phosphate method, lipofection or electroporation.
[0043] By culturing the thus obtained transformant in a medium, the
GnT-IV protein is produced.
[0044] In the cultivation of a transformant, any medium may be used
as long as the host is viable in it. For example, LB medium or the
like may be used if the host is E. coli. If the host is yeast, YPD
medium or the like may be used. If the host is an animal cell,
Dulbecco's medium supplemented with an animal serum or the like may
be used. The cultivation is performed under conditions
conventionally used for the host. For example, if the host is E.
coli, cells are cultured at about 30-37.degree. C. for about 3-24
hours with, if necessary, aeration and/or agitation. If the host is
yeast, cells are cultured at about 25-37.degree. C. for about 12
hours to 2 weeks with, if necessary, aeration and/or agitation. If
the host is an animal cell, cultivation is performed at about
32-37.degree. C. under 5% CO.sub.2 and 100% humidity for about 24
hours to 2 weeks with, if necessary, change of the aeration
conditions and/or agitation.
[0045] After the cultivation, the cultured microorganism or cells
are disrupted using a homogenizer, French press, sonication,
lysozyme and/or freeze-thawing to thereby elute the GnT-IV protein
outside the microorganism or cells. Then, the protein can be
obtained from soluble fractions. If the protein of interest is
contained in insoluble fractions, the insoluble fractions are
collected by centrifugation after disruption of the microorganism
or cells. Then, the protein may be solubilized with a buffer
containing guanidine hydrochloride or the like for recovery.
Alternatively, the cultured microorganism or cells may be disrupted
directly with a buffer containing a protein denaturing agent such
as guanidine hydrochloride to thereby elute the protein of interest
outside the microorganism or cells.
[0046] Purification of the GnT-IV protein from the above
supernatant may be performed by the method described in Example 1.
Alternatively, this purification may be performed by appropriately
combining conventional separation/purification methods. These
conventional separation/purification methods include, but are not
limited to, centrifugation, salting out, solvent precipitation,
dialysis, ultrafiltration, partition chromatography, gel
filtration, capillary electrophoresis, TLC, ion exchange
chromatography, metal chelate chromatography, affinity
chromatography, reversed phase chromatography and isoelectric
focusing.
[0047] The biochemical properties of the GnT-IV enzyme protein
obtained from bovine small intestine as described above are as
follows.
[0048] (1) Action
[0049] This enzyme protein produces a saccharide having a partial
structure represented by the formula below: 4
[0050] using UDP-GlcNAc as a sugar donor and a saccharide having a
partial structure represented by the formula below as a sugar
receptor: 5
[0051] The saccharide as a sugar receptor means an oligosaccharide,
polysaccharide, glycoconjugate (glycopeptide, glycoprotein,
glycolipid or proteoglycan) or a derivative thereof.
[0052] (2) Substrate Specificity
[0053] When the sugar receptor is an oligosaccharide (for the
structures of oligosaccharides, see FIG. 4), the enzyme protein
exhibits reactivities of 0% toward core type oligosaccharides, 54%
toward GnT-I product type oligosaccharides and 164% toward GnT-V
product type oligosaccharides, wherein the reactivity of the enzyme
protein toward GnT-II product type oligosaccharides is regarded as
100%.
[0054] The enzyme protein exhibits a reactivity of 46% toward a
structure of GnT-II product type oligosaccharides in which fucose
is attached via .alpha.1.fwdarw.6 linkage to the GlcNAc at the
reducing terminus.
[0055] The enzyme protein exhibits a reactivity of 0% toward a
structure of GnT-II product type oligosaccharides in which the
GlcNAc on the a 1.fwdarw.3 mannose is lacking.
[0056] The enzyme protein exhibits a reactivity of 16% toward a
structure of GnT-II product type oligosaccharides in which
galactose is attached via .beta.1.fwdarw.4 linkage to the GlcNAc on
the .alpha.1.fwdarw.6 mannose, and a reactivity of 0% toward a
structure of GnT-II product type oligosaccharides in which
galactose is attached via .beta.1.fwdarw.4 linkage to the GlcNAc on
the .alpha.1.fwdarw.3 mannose.
[0057] The enzyme protein exhibits a reactivity of 0% toward a
structure of GnT-II product type oligosaccharides in which GlcNAc
is attached via .beta.1.fwdarw.4 linkage to the .beta.1.fwdarw.4
mannose.
[0058] (3) Molecular Weight
[0059] About 66K as determined by SDS-PAGE (under non-reducing
conditions). About 60K after treatment with peptide N-glycosidase
F. Since a shift of band is observed when peptide N-glycanase is
used, the enzyme protein is thought to be a glycoprotein.
[0060] The apparent molecular weight as determined by filtration
with a gel containing Triton X-100 is 77K. Thus, it is thought that
GnT-IV does not have a subunit structure and functions as a
monomer.
[0061] The protein moiety of this enzyme deduced from the
nucleotide sequence thereof consists of 535 amino acid residues and
has a molecular weight of 61614.
[0062] (4) Optimum pH
[0063] The optimum pH for reaction is about 5.5. More than 50% of
the maximum activity is observed in the range from pH 6.5 to
8.0.
[0064] (5) Inhibition, Activation and Stabilization
[0065] (i) Inhibition
[0066] The activity of this enzyme is inhibited by addition of 20
mM EDTA.
[0067] This enzyme is inhibited by UDP derivatives. The intensity
of inhibition is in the following order:
UDP>>UDP-Glc>UDP-GalNAc>- ;>2'-deoxy
UDP>UDP-hexanolamine>>UDP-Gal>UTP>UDP-glucuro- nic
acid>UMP.
[0068] Uridine, TDP and CDP do not have inhibitory effect.
[0069] (ii) Activation
[0070] A divalent cation is essential for expression of the
activity. Among divalent cations, Mn.sup.2+ shows the greatest
effect. At a concentration of 7.5 mM, the respective effects of
Co.sup.2+ and Mg.sup.2+ are about 70% of that of Mn.sup.2+, and the
effect of Ca.sup.2+ is about 10% of that of Mn.sup.2+. The effect
of Mn.sup.2+ is greatest in the range from 5 to 20 mM.
[0071] (iii) Stabilization
[0072] Stabilizing effect is recognized in BSA and glycerol.
[0073] (6) Kinetic Parameters
[0074] When the saccharide as a receptor is an oligosaccharide (for
the structures of oligosaccharide, see FIG. 4):
[0075] (i) under assay conditions in which the enzyme is reacted in
50 .mu.l of 125 mM MOPS buffer (pH 7.3) containing 0.8 mM receptor
substrate, 20 mM UDP-GlcNAc, 7.5 mM MnCl.sub.2, 200 mM GlcNAc, 0.5%
(w/v) Triton X-100, 10% glycerol and 1% BSA at 37.degree. C. for 4
hours:
[0076] Km and Vmax values toward GnT-II product type
oligosaccharide are 0.73 mM and 3.23 .mu.M/min, respectively.
[0077] Km and Vmax values toward GnT-V product type oligosaccharide
are 0.13 mM and 1.75 .mu.M/min, respectively.
[0078] When GnT-II product type oligosaccharide is the receptor
substrate, Km value toward UDP-GlcNAc is 0.22 mM.
[0079] (ii) under assay conditions in which the enzyme is reacted
in 125 mM MOPS buffer (pH 7.3) containing 120 mM UDP-GlcNAc, 7.5 nM
MnCl.sub.2, 0.5% (w/v) Triton X-100, 10% glycerol and 1% BSA at
37.degree. C. for 4 hours:
[0080] Km and Vmax values toward GnT-II product type
oligosaccharide are 0.59 mM and 0.74 mM/min/mg, respectively.
[0081] Km and Vmax values toward GnT-V product type oligosaccharide
are 0.14 mM and 0.47 mM/min/mg, respectively.
[0082] (7) GnT-IV Family
[0083] The homology between bovine GnT-IVa and human GnT-IVa is 91%
at the nucleic acid level and 96% at the amino acid level.
[0084] All of the partial amino acid structures contained in the
purified GnT-IV from bovine small intestine are encoded in the
bovine GnT-IVa gene.
[0085] Human GnT-IVb and human GnT-IVa have 63% homology at the
nucleic acid level and 62% homology at the amino acid level.
However, they are entirely different in the C-terminal and
N-terminal regions.
[0086] From the biochemical properties described above, the GnT-IV
of the invention has been recognized as a novel enzyme in the point
that this enzyme is able to perform the following reaction which
conventional enzymes cannot perform: 6
BRIEF DESCRIPTION OF THE DRAWINGS
[0087] FIG. 1 shows the biosynthetic pathway of Asn-linked sugar
chains.
[0088] FIG. 2 shows variations of the Asn-linked sugar chain
(revised from FIG. 1 in Makoto Takeuchi, Wako Purechemical
Newsletter 64, 18-19, 1996).
[0089] a. Mannan type: a sugar chain structure characteristic of
fungi such as yeasts and molds.
[0090] b. Xylo-high-mannose type: a structure characteristic of
plants, mollusks and insects.
[0091] c. High mannose type: a structure commonly seen in plants,
insects and animals.
[0092] d. Hybrid type: a structure commonly seen in insects and
animals.
[0093] e. Complex type: a structure characteristic of animals.
[0094] f. Prokaryotic cells: have no system for biosynthesis of
Asn-linked sugar chains.
[0095] The portion boxed with dotted lines represents the common
core sugar chain.
[0096] FIG. 3 shows the positions of GlcNAc transfer by various
GlcNAc transferases (GlcNAc glycosyltransferases).
[0097] FIG. 4 shows the designations and structures of
oligosaccharides.
[0098] FIG. 5 shows a high performance liquid chromatogram for
GnT-IV reaction products.
[0099] FIG. 6 shows the results of analysis of GnT-IV by
Q-Sepharose FF chromatography.
[0100] FIG. 7 show the results of analysis of GnT-IV by copper
chelate Sepharose FF chromatography.
[0101] FIG. 8 shows the results of analysis of GnT-IV by
UDP-Hexanolamine Agarose affinity chromatography (I).
[0102] FIG. 9 shows the results of analysis of GnT-IV by
UDP-Hexanolamine Agarose affinity chromatography (II).
[0103] FIG. 10 shows the results of analysis of GnT-IV by Superdex
200 gel chromatography.
[0104] FIG. 11 is a photograph showing the results of SDS-PAGE (SDS
polyacrylamide gel electrophoresis) of purified GnT-IV.
[0105] FIG. 12 shows the results of native gel electrophoresis of
purified GnT-IV and the activity thereof.
[0106] FIG. 13 shows Smith degradation profile of GnT-IV, -V and VI
product type oligosaccharides.
[0107] FIG. 14 shows the results of .sup.1H-NMR (30.degree. C.) of
the GnT-IV reaction product.
[0108] FIG. 15 shows the optimum pH for GnT-IV.
[0109] FIG. 16 shows the optimum Mn2+ concentration for GnT-IV.
[0110] FIG. 17 shows the results of analysis by SDS-PAGE and
fluorochromatography of glycoproteins which are the reaction
products of GnT-IV.
[0111] Lanes 1 and 2: 7.6 .mu.g of asialo agalacto human
transferrin
[0112] Lane 3: 7.6 .mu.g of asialo human transferrin
[0113] Lanes 4 and 5: 2.8 .mu.g of asialo agalacto, CHO
cell-derived recombinant human erythropoietin
[0114] Lanes 6 and 7: 1.3 .mu.g of asialo agalacto fetuin
[0115] Lanes 1, 4 and 6 represent mock experiments in which
reaction was performed without GnT-IV. M represents molecular
markers (Bio-Rad). PM represents pre-stained molecular markers
(Bio-Rad, USA).
[0116] GnT-IV reaction conditions: To 10 .mu.l of a solution
containing 0.702 mnol/hr of GnT-IV, a substrate glycoprotein
equivalent to 1.6 nmol of biantennary type sugar chains (for fetuin
alone, the sugar chain content was 1.6 nmol) and 450 nCi of
UDP-[.sup.14C] GlcNAc, an equal volume of an assay mixture (250 mM
MOPS buffer, pH 7.3, 400 mM GlcNAc, 20% glycerol, 1.0% (w/v) Triton
X-100, 15 mM MnCl.sub.2, 1 mM UDP-GlcNAc) was added to obtain a
reaction solution, which was incubated at 37.degree. C. for 20
hours. One tenth of the resultant solution was analyzed by SDS-PAGE
and fluorography.
[0117] For SDS-PAGE, 10-20% gradient gel (Daiichi Kagaku) was used.
For fluorography, Amplify (Amersham) was used to expose the samples
to X ray film for 20 hours. The visualization of biantennary sugar
chains in the glycoprotein was performed using ConA-HRP and
POD-Immunostain Kit (Wako Purechemical) after dot-blotting onto a
PVDF membrane.
[0118] FIG. 18 shows the open reading frame of human GnT-IVa and
the region contained in pCore-His expression vector.
[0119] FIG. 19 shows the results of isoelectric focusing and
Western analysis of erythropoietins produced by individual cell
clones. Using two erythropoietin-producing strains and the same
strains into which bovine and human GnT-IVa genes were introduced,
respectively, the erythropoietin secreted by each strain was
analyzed by isoelectric focusing and Western blotting using
anti-erythropoietin antibody. On the left side, the positions of pI
markers are shown.
BEST MODES FOR CARRYING OUT THE INVENTION
[0120] Hereinbelow, the present invention will be described in more
detail with reference to the following Examples. However, the
present invention is not limited to these Examples.
REFERENCE EXAMPLE 1
[0121] (1) Reagents Used in the Examples
[0122] Unless indicated otherwise, the reagents used were the
highest grade products manufactured by Wako Purechemical
Industries, Ltd.
[0123] (i) Pyridylaminated Oligosaccharides
[0124] Each of the pyridylaminated oligosaccharides used was
obtained as described below. First, pyridylaminated
oligosaccharides were prepared from human transferrin (apo type;
Sigma, USA) according to the method of Tokugawa et al. [Biehuizen,
M. F., Mattel, M. G. and Fukuda, M. (1993) Genes Dev., 7, 468-478].
The resultant material was treated with one or a combination of the
following enzymes: Arethrobacter ureafaciens-derived sialidase
(Nacalai Tesque), Asperugillus sp.-derived .beta.-galactosidase
(Toyobo), Jack bean-derived .beta.-N-acetylhexosaminidase
(Seikagaku Corp.), GnT-V active fraction in CHO-K1 cell extract
(supernatant obtained by sonication of CHO-K1 cells in 2 volumes of
5 mM Tris-HCl buffer, pH 7.5, containing 2 mM MgCl.sub.2 and 1 mM
PMSF, and then centrifugation at 900.times.g for 10 min), and the
GnT-V active fraction in the solubilized fraction from bovine small
intestine homogenate (for the method of preparation, see
"Preparation of the Microsome Fraction" and "Solubilization" in
Example 1). A part of the pyridylaminated oligosaccharides were
prepared by treating PA-Sugar Chain 021 and 022 (Takara Shuzo) with
the above enzymes. In both cases, the oligosaccharides prepared
were purified by reversed phase chromatography using an ODS column
(10.times.250 mm; Vydac, USA) before use.
[0125] (ii) Glycoprotein Substrates
[0126] Bovine fetuin (Sigma, USA) and CHO cell-derived recombinant
human erythropoietin (Kirin Brewery) were subjected to the
following pretreatment to purify them into relatively uniform
glycoform. Briefly, 40-100 mg of the glycoprotein was applied to a
ConA-Sepharose column (5 ml; Pharmacia, Sweden) equilibrated with
10 mM Tris-HCl buffer, pH 7.4, containing 1 mM MgCl.sub.2, 1 mM
CaCl.sub.2 and 0.15 M NaCl to thereby obtain a glycoform with a low
biantennary sugar chain content as the non-adsorbed fraction.
Thereafter, the column was eluted with the above buffer containing
1.0 M .alpha.-methyl mannoside (Nacalai Tesque) to thereby obtain
the fraction adsorbing a glycoform with a high biantennary sugar
chain content. Thus, fetuin with a low biantennary sugar chain
content and erythropoietin with a high biantennary sugar chain
content were obtained. With respect to human transferrin, there is
no need to purify this glycoprotein since almost all sugar chains
thereof are biantennary.
[0127] The thus obtained fetuin and human transferrin were
individually reacted with 1 U of sialidase and 0 or 107 U of
.beta.-galactosidase in 1 ml of 0.4 M sodium acetate buffer, pH
5.0, containing 4 mM MgCl.sub.2 at 37.degree. C. for 16 hours to
thereby obtain asialo or asialo agalacto glycoproteins. The
erythropoietin with a high biantennary sugar chain content was
reacted with 0.5 U of sialidase and 5 U of .beta.-galactosidase in
the same manner as described above to obtain an asialo agalacto
glycoprotein.
[0128] Each of the thus obtained glycoprotein substrates was
dialyzed against 50 mM ammonium acetate buffer, pH 7.3. Then, the
amount of protein was determined with BCA protein assay (Pierce,
USA) using BSA (bovine serum albumin) as a standard. Further, the
protein was analyzed by SDS-PAGE (sodium dodesyl sulfate
polyacrylamide gel electrophoresis). The thus prepared
glycoproteins were used in the Examples.
[0129] (iii) RT-PCR (Reverse Transcription-Polymerase Chain
Reaction)
[0130] For RT-PCR, Access RT-PCR System (Promega, USA) was used.
For the amplification of a fragment of a gene of interest, Pfu
polymerase (Stratagene, USA) was used.
[0131] (2) Equipment used in the Examples
[0132] (i) Gene Sequencing
[0133] ABI PLISM 377 DNA Sequencer (Perkin-Elmer, USA) was
used.
REFERENCE EXAMPLE 2
Specific Assay for GnT-IV Activity
[0134] Generally, there are two methods for assaying GnT-IV
activity: a method in which the transfer of radiolabeled GlcNAc to
an oligosaccharide substrate is examined and a method in which the
transfer of GlcNAc to a labeled oligosaccharide substrate is
fractionally analyzed by HPLC or the like. Taniguchi et al.
developed a method in which GnT-III, -IV and -V activities are
simultaneously determined using the GnT-II product type
oligosaccharide as a receptor [Nishikawa, A., Fujii, S., Sugiyama,
T. and Taniguchi, N. (1988) Anal. Biochem., 170, 349-354]. However,
this assay method as it is was inappropriate for the assay during
purification of GnT-IV because the relative activity of GnT-IV is
much lower than those of GnT-III and -V.
[0135] Then, the present inventors have developed a method for
determining GnT-IV activity quantitatively and sensitively by
increasing the amount of the acceptor pyridylaminated
oligosaccharide to 10-fold compared to the amount used in the
previous assay system [Tokugawa, K., Oguri, S. and Takeuchi, M.
(1996) Glycoconjugate J., 13, 53-56]. Generally, it is very
difficult to prepare such a large amount of acceptor
oligosaccharides. However, according to the method of Tokugawa et
al. [Tokugawa, K., Oguri, S. and Takeuchi, M. (1996) Glycoconjugate
J., 13, 53-56], such oligosacchardes are readily prepared.
[0136] In Examples of the present invention, GnT-IV activity was
assayed as described below.
[0137] The enzyme was reacted in 125 mM MOPS
[3-(N-morpholino)propane-sulf- onic acid] buffer, pH 7.3,
containing 0.8 mM pyridylaminated oligosaccharide substrate (GNT-II
product type oligosaccharide substrate), 20 mM UDP-GlcNAc, 7.5 mM
MnCl.sub.2, 200 mM GlcNAc, 0.5% (w/v) Triton X-100, 10% glycerol
and 1% BSA at 37.degree. C. for 4 hours. Then, the reaction was
terminated by boiling the solution for 2 minutes. After removal of
solids with a 0.45 nm filter, 5 .mu.l of the filtrate was analyzed
with an ODS-80TM column (4.6.times.150 mm; TOSO) (FIG. 5) at
50.degree. C. with 50 mM ammonium acetate buffer, pH 4.0,
containing 0.15% (w/v) n-butanol at a flow rate of 1.2 ml/min. The
fluorescence of pyridylamino groups was detected using excitation
at 320 nm and emission at 400 nm.
EXAMPLE 1
Isolation and Purification of the Enzyme
[0138] (1) Screening of a Source of the Enzyme
[0139] A source of the GnT-IV enzyme to be purified was searched
for by utilizing the assay method described above. It was found
that the relative activity of GnT-IV to those of GnT-III and GnT-V
in bovine small intestine is rather higher than the relative
activities of GnT-IV in any other tissues as shown in Table 1.
Thus, bovine small intestine was selected as a starting material
for purification.
1TABLE 1 Search for Sources of GnT-IV Enzyme Specific Activity
(pmol/h .multidot. mg-protein) Source of the Enzyme IV III V
Cultured cells CHO 10.8 0 1097 Bowes 12.0 341 150 AH66.sup.1) 2.0
634 30 Solid AH.sup.1) 27 116 80 Yoshida sarcoma.sup.1) 1.3 70 109
Rat organs.sup.1) Small intestine 17 280 68 Heart 9.4 11 10 Spleen
20 100 21 Kidney 1.9 1840 30 Brain 3.7 660 38 Human.sup.1) Liver
2.8 8.1 8.2 Bovine organs Small intestine 25 174 41 Heart N.D. N.D.
N.D. Spleen 10.9 0.7 10.9 Colostrum N.D. N.D. N.D. N.D.: below
detection limit .sup.1): data from Nishikawa, A. et al., BBA 1035,
313-318 (1990)
[0140] (2) Purification
[0141] Unless otherwise indicated, all the operations were
performed at 4.degree. C.
[0142] (i) Preparation of the Microsome Fraction
[0143] Two kilograms of bovine small intestine (obtained from a
meat processor) was minced. Then, 4 volumes of an extraction buffer
(10 mM Tris-HCl buffer, pH7.4, containing 0.25 M sucrose, 1 mM
phenylmethylsulfonyl fluoride, 1 mM benzamidine hydrochloride, 1 mM
dithiothreitol and 10 mg/ml antipain) was added thereto and
homogenized with Polytron (Kinematica, Sweden). The resultant
homogenate was centrifuged at 900.times.g for 10 minutes. Then, the
supernatant was centrifuged further at 105,000.times.g for 60
minutes to thereby obtain the microsome fraction as a precipitate
(Sample 1).
[0144] (ii) Solubilization
[0145] Sample 1 was suspended in 3 volumes of a solubilization
buffer prepared by adding Triton-100 to the extraction buffer to
give a final concentration of 1%. The supernatant was obtained by
the centrifugation at 105,000.times.g for 60 minutes. The pellet
was suspended again and collect the supernatant. The first and
second extracts were combined (Sample 2).
[0146] (iii) Q-Sepharose FF Chromatography
[0147] Sample 2 was applied to Q-Sepharose FF Column (5.times.30
cm; Pharmacia, Sweden) pre-equilibrated with operation buffer 1 (20
mM Tris-HCl, pH 7.4, containing 1 mM benzamidine hydrochloride,
0.1% Triton X-100 and 20% glycerol) and then eluted by a linear
gradient of 0-0.5 M KCl (FIG. 6) (Sample 3).
[0148] (iv) Copper Chelate Sepharose FF Chromatography
[0149] Sample 3 was applied to Copper Chelate Sepharose FF Column
(5.times.10 cm; Pharmacia, Sweden) pre-equilibrated with operation
buffer 2 (obtainable by adding KCl to operation buffer 1 at a final
concentration of 0.15 M). Then, non-adsorbed fractions were washed
out with 5 volumes of operation buffer 2. Thereafter, the adsorbate
was eluted by a linear gradient of 0.01 M glycine (FIG. 7). The
resultant GnT-IV active fraction was pooled and concentrated with
YM30 ultrafiltration membrane (Amicon, USA) (Sample 4).
[0150] (v) UDP-Hexanolamine Agarose Affinity Chromatography I
[0151] To UDP-Hexanolamine Agarose Affinity Column (1.2.times.4.5
cm; Sigma, USA) pre-equilibrated with operation buffer 3 (20 mM
Tris-HCl, pH 8.0, containing 0.15 M KCl, 10 mM MnCl.sub.2, 0.05%
Triton X-100 and 20% glycerol), one half of Sample 4 dialyzed
against 1 mM benzamidine hydrochloride-added operation buffer 3 was
applied. Then, non-adsorbed fractions were washed out with
operation buffer 4 (20 mM Tris-HCl, pH 8.0, containing 10 mM
MnCl.sub.2, 0.05% Triton X-100 and 20% glycerol). Thereafter, the
adsorbate was eluted with operation buffer 4 to which 1 M (final
concentration) KC1 had been added (FIG. 8). The GnT-IV active
fraction was pooled and dialyzed against operation buffer 5 (having
the same composition as that of operation buffer 4 but having a pH
of 7.4) (Sample 5).
[0152] (vi) UDP-Hexanolamine Agarose Affinity Chromatography II
[0153] Sample 5 was applied to UDP-Hexanolamine Agarose Affinity
Column (1.0.times.6.5 cm; Sigma, USA) pre-equilibrated with
operation buffer 5. Then, non-adsorbed fractions were washed out
with operation buffer 5. Thereafter, the adsorbate was eluted with
MnCl.sub.2-removed operation buffer 5 (FIG. 9). The resultant
GnT-IV active fraction was pooled (Sample 6).
[0154] (vii) Superdex 200 Gel Chromatography
[0155] Sample 6 was concentrated with a small Q-sepharose FF column
and applied to Superdex 200HR5/5 Column (1.times.30 cm; Pharmacia,
Sweden) pre-equilibrated with operation buffer 6 (obtained by
adding KCl to operation buffer 5 at a final concentration of 0.15
M) (FIG. 10). Operation buffer 6 was applied to the column at a
flow rate of 0.25 ml/min to thereby obtain the GnT-IV active
fraction (Sample 7).
[0156] (viii) The amount of protein, activity and specific activity
in each purification step are summarized in Table 2. The final
sample was purified 224,000-fold compared to the small intestine
homogenate.
2TABLE 2 Purification of GnT-IV Amount of Total enzyme Specific
activity Yield Purification Purification Step protein (mg) activity
(nmol/h) (nmol/h/mg) (%) factor (-fold) Bovine small intesting
112,900 49,500 0.44 100 1 homogenate Solubilized fraction 24,100
14,500 0.60 29 1.4 Q-Sepharose 4,000 7,200 1.80 14 4.1 Cu Chelate
Sepharose 450 3,670 8.10 7.4 18.4 UDP-Hexanolamine I 0.59 1,950
3,310 3.9 7,510 UDP-Hexanolamine II 0.035 1,420 40,600 2.9 92,200
Superdex 200 0.008 790 98,800 1.6 224,000 Started from 2 kg of
bovine small intestine.
[0157] (3) Properties in terms of Enzyme Chemistry and Protein
Chemistry
[0158] (i) Purity
[0159] Sample 7 gave a single band at a molecular weight of 60K in
SDS-PAGE (FIG. 11). When Sample 7 was subjected to native-PAGE and
the resultant band was cut out from the gel to determine GnT-IV
activity, the location of the protein band agreed with the location
of the activity (FIG. 12). Furthermore, any of GnT-I, -II -III or
-V activity was not detected in Sample 7. From these findings, it
was concluded that Sample 7 is pure GnT-IV. Taking into account
that the apparent molecular weight of this protein was 77K as
determined by Triton X-100-containing gel filtration (FIG. 10), it
is thought that GnT-IV does not have a subunit structure and
expresses its activity in the form of a monomer. When Sample 7 was
treated with Peptide N-Glycosidase F (Boehringer-Mannheim,
Germany), an increase in mobility was observed on SDS-PAGE. Thus,
it is thought that GnT-IV from bovine small intestine is a
glycoprotein having at least Asn-linked sugar chains.
[0160] (ii) Reaction Specificity
[0161] When this enzyme reacts on the GnT-II product type
oligosaccharide represented by the formula below as a substrate:
7
[0162] under the standard assay conditions, the enzyme yielded a
single product (pyridylaminated oligosaccharide 1) as assayed by
HPLC.
[0163] This product was collected, followed by determination of its
structure by (i) a combination of Smith degradation and laser
TOF-MS (time-of-flight mass spectrometer) and (ii) .sup.1H-NMR.
Thus, the reaction specificity of this enzyme was examined. When
pyridylaminated oligosaccharide 1 was subjected to Smith
degradation according to the method of Kobata and Takasaki [Kobata,
A. and Takasaki, S. (1993) in Glycobiology "A Practical Approach"
(Fukuda, M. and Kobata, A., eds) 165-185, IRL Press, Oxford,
England], its mass number changed from 1599.0 to 795.30 as a result
of the first degradation and further changed to 634.68 as a result
of the second degradation. This agrees to the reaction pathway as
shown in FIG. 13. Thus, it was concluded that the reaction product
of this enzyme has the following structure: 8
[0164] Further, when pyridylaminated oligosaccharide 1 was
subjected to .sup.1H-NMR, a peak of 4.53 ppm which corresponds to
the anomeric proton of GlcNAc7 shown in the following formula was
detected; its coupling constant J1,2 was 7.9 Hz (FIG. 14). These
results indicate that GlcNAc7 is, as shown in the formula below,
attached to position 4 of Man4 via .beta.-type linkage, supporting
the above structure completely. 9
[0165] (iii) Optimum pH
[0166] The optimum pH of this enzyme is around 7.5 as shown in FIG.
15.
[0167] (iv) Requirement of Divalent Cation
[0168] As shown in Table 3, this enzyme is deactivated by the
addition of EDTA (ethylenediamine tetra-acetic acid). A divalent
cation is essential for its activity. Among divalent cations,
Mn.sup.2+ shows the greatest effect, followed by Co.sup.2+ and
Mg.sup.2+. Weak effect is recognized in Ca.sup.2+ and Fe.sup.2+.
The optimum concentration of Mn.sup.2+ is around 10 mM as shown in
FIG. 16.
3TABLE 3 Divalent Cation Requirement of GnT-IV Additive Activity
(%) None 5.6 EDTA 0 MnCl.sub.2 100 CoCl.sub.2 74.8 MgCl.sub.2 72.5
CaCl.sub.2 7.2 FeCl.sub.2 9.8 CuCl.sub.2 0
[0169] GnT-IV activity was determined by adding each of the metal
ions (10 mM) to a GnT-IV sample from which metal ions had been
removed. GnT-IV activity is represented in per cent in the Table,
wherein the activity when 10 mM MnCl.sub.2 was added is regarded as
100%.
[0170] (v) Inhibition by Sugar Nucleotides
[0171] As shown in Table 4, UDP inhibited the activity of this
enzyme most strongly. The inhibitory effects of UDP-glucose,
UDP-GalNAc, 2'-deoxy-UDP and UDP-hexanolamine (Sigma, USA) followed
that of UDP in this order. Uridine, UMP, TDP and CDP exhibited
little inhibitory effect.
4TABLE 4 Inhibition of GnT-IV by Nucleotides Additive Activity (%)
None 100 Uridine 115 UMP 97.3 UDP 27.3 UTP 88.2 TDP 110 CDP 112
2'-deoxy-UDP 67.4 UDP-hexanolamine 73.6 UDP-glucose 56.6
UDP-galactose 87.3 UDP-glucuronic acid 92.3
UDP-N-acetylgalactosamine 59.7
[0172] GnT-IV activity when each nucleotide (2 mM) was added in the
presence of 0.5 mM UDP-GlcNAc is expressed in per cent in the
Table, wherein the activity when nothing was added is regarded as
100%.
[0173] (vi) Substrate Specificity
[0174] As shown in Table 5, this enzyme preferred the GnT-V product
type oligosaccharide (E in Table 5) the most as an acceptor. Next
to this, the enzyme preferred the GnT-II product type
oligosaccharides (D in Table 5).
[0175] When the reactivity of this enzyme toward the GnT-II type
oligosaccharide is regarded as 100%, this enzyme exhibits
reactivities of 0% and 54% toward the core type oligosaccharides (A
in Table 5) and the GnT-I product type oligosaccharides (C in Table
5), respectively.
[0176] This enzyme exhibits a reactivity of 46% toward a structure
of GnT-II product type oligosaccharide in which fucose is attached
via .alpha.1.fwdarw.6 linkage to the GlcNAc at the reducing
terminus (F in Table 5).
[0177] This enzyme exhibits a reactivity of 0% toward a structure
of GnT-II product type oligosaccharides in which the GlcNAc on the
.alpha.1.fwdarw.3 mannose is lacking (B in Table 5).
[0178] This enzyme exhibits a reactivity of 16% toward a structure
of GnT-II product type oligosaccharides in which galactose is
attached via .beta.1.fwdarw.4 linkage to the GlcNAc on the
.alpha.1.fwdarw.6 mannose (G in Table 5), and a reactivity of 0%
toward a structure of GnT-II product type oligosaccharide in which
galactose is attached via .beta.1.fwdarw.4 linkage to the GlcNAc on
the .alpha.1.fwdarw.3 mannose (H and I in Table 5).
[0179] This enzyme exhibits a reactivity of 0% toward a structure
of GnT-II product type oligosaccharides in which GlcNAc is attached
via .beta.1.fwdarw.4 linkage to the .beta.1.fwdarw.4 mannose (J in
Table 5).
[0180] The substrate specificity of this enzyme as described above
almost agree with the substrate specificity of GnT-IV predicted by
Schachter et al. [Glesson, P. A. and Schachter, H. (1983) J. Biol.
Chem., 258, 6162-6173]. Thus, it has become clear that this enzyme
of the invention is the very GnT-IV that has long been a missing
link in the biosynthesis of complex type sugar chains.
5 TABLE 5 Relative Activity of Receptor Oligosaccharide GlcNAc
Transfer (%) A 10 0 B 11 0 C 12 54 D 13 100 E 14 164 F 15 46 G 16
16 H 17 0 I 18 0 J 19 0
[0181] (vii) Kinetic Parameters
[0182] Under the assay conditions as described in Reference Example
2, the Km and Vmax values of this enzyme toward the GnT-II product
type oligosaccharides were 0.73 mM and 3.23 .mu.M/min,
respectively, and these values toward the GnT-V product type
oligosaccharides were 0.13 mM and 1.75 .mu.M/min, respectively. The
Km value toward the UDP-GlcNAc was 0.22 mM.
[0183] Among the pyridylaminated oligosaccharides obtained, those
represented by the following formulas were found to be novel
oligosaccharides: 20
[0184] and 21
[0185] (viii) Action on Glycoproteins
[0186] In order to demonstrate that GnT-IV can act on not only
oligosaccharide substrates but also oligosaccharide chains on
glycoproteins, GnT-IV reacts on asialo agalacto glycoproteins using
UDP-(.sup.14C] GlcNAc as a sugar donor. Then, the reaction products
were analyzed by SDS-PAGE and fluorography (Panels A and B, FIG.
17). As shown in lanes 2 and 5 of Panel B in FIG. 17, transfer of
[.sup.14C] GlcNAc to asialo agalacto human transferrin and asialo
agalacto, CHO cell-derived recombinant human erythropoietin.
[0187] The human transferrin having the GnT-IV product type sugar
chain (of the following formula) obtained by this GnT-IV reaction
is a novel substance which does not occur in nature. 22
EXAMPLE 2
Peptide Mapping Analysis
[0188] About 1 mg of this enzyme of the invention finally purified
was electrophoresed on 0.1% SDS-10% polyacrylamide gel according to
the method of Laemmli [Laemmli, U. K. Nature (1970) 313, 756-762].
The separated proteins were electroblotted onto a PVDF membrane.
The protein fixed on the membrane was S-carboxymethylated and then
digested with lysylendopeptidase Achromobacter protease I (AP-I)
(Wako Pure Chemical Industries, Ltd) to obtain an AP-I-digested
fragment mixture. The AP-I-digested PVDF membrane was further
digested with Asp-N (Takara Shuzo) to obtain an Asp-N-digested
fragment mixture. Each of the peptide fragment mixtures was
separated by high performance liquid chromatography and subjected
to amino acid sequence analysis. As a result, the sequences shown
in SEQ ID NOS: 1-14 were obtained.
EXAMPLE 3
Isolation and Identification of Bovine GnT-IVa cDNA
[0189] (1) RT-PCR
[0190] Based on the amino acid sequences shown in SEQ ID NOS: 7 and
11 obtained in Example 2, oligomer AP-5F shown in SEQ ID NO: 15 and
oligomer DN-9R shown in SEQ ID NO: 16 were synthesized
respectively. An RT-PCR was performed using as a template the total
RNA extracted from bovine small intestine tissue by the guanidium
isothiocyanate method and using the above primers. As a result, an
amplified fragment of about 170 bp which seemed specific was
obtained. This fragment was subcloned.
[0191] (2) Screening of a Library
[0192] A bovine small intestine cDNA library (Clontech, USA) was
screened with the above-mentioned RT-PCR product to obtain four
positive plaques. The nucleotide sequences of these clones were
determined. The resultant sequences contained a number of
nucleotide sequences coding for some of the partial amino acid
sequences (SEQ ID NOS: 1-14) obtained in Example 2, and also
contained a sequence appearing to be a termination codon. Using a
fragment of 150 bp representing the most upstream region of the
resultant nucleotide sequence, the library was screened again to
obtain two positive plaques. The nucleotide sequences of these
clones were determined. Then, the library was further screened
similarly with a probe of 150 bp of the most upstream region,
however, new clones were not obtained.
[0193] (3) 5' RACE (Rapid Amplification of the cDNA Ends)
[0194] Subsequently, 5' RACE was performed in order to obtain a
full-length cDNA. Using the sequence of the most upstream region
obtained by the phage screening, the first 5' RACE was performed.
However, an initiation codon could not be found. Then, based on the
sequence obtained by the first 5' RACE, the second 5' RACE was
performed to thereby obtain a sequence containing an initiation
codon. This sequence was ligated to the previously obtained partial
gene sequence of the phage clone to thereby obtain a gene fragment
containing an intact open reading frame (Gene 1). The nucleotide
sequence for the thus obtained gene fragment is shown in SEQ ID NO:
17, and the amino acid sequence deduced therefrom is shown in SEQ
ID NO: 18. It was confirmed that this DNA fragment contains all of
the nucleotide sequences coding for the 14 partial amino acid
sequences (SEQ ID NOS: 1-14) obtained in Example 2.
EXAMPLE 4
Construction of an Expression Vector Using the Cloned Bovine
GnT-IVa Gene and a Method for Producing GnT-IVa Enzyme
[0195] (1) Construction of a Vector
[0196] A primer (SEQ ID NO: 19) which introduces an XhoI site into
a region upstream of the initiation codon of Gene 1 and another
primer (SEQ ID NO: 20) which introduces an XbaI site into a region
downstream of the termination codon of the gene were synthesized.
Then, the entire gene coding for GnT-IV enzyme was amplified by PCR
with the primers. The amplified fragment obtained was digested with
XhoI and XbaI, and inserted between the XhoI and XbaI sites of pSVL
vector (Pharmacia, Sweden) to prepare plasmid pBGT4.
[0197] (2) Introduction into COS7 Cells
[0198] Plasmid pBGT4 was introduced into COS7 cells (RIKEN Cell
Bank) by electroporation. Briefly, 10 .mu.g of the plasmid was
added to approximately 5.times.10.sup.6 cells in 0.8 ml of PBS(-)
(Nissui Pharmaceutical Co.). A voltage of 1600 V was applied at a
capacitance of 25 .mu.F with a gene pulser (BioRad, USA) at room
temperature to introduce the gene into the cells. The resultant
cells were transferred to a 90 mm laboratory dish and cultured in
10 ml of Dulbecco's modified Eagle's medium (Base Catalogue No.
12430, Life Technologies, Inc., USA) containing 10% fetal bovine
serum under 5% CO.sub.2 at 37.degree. C. for 72 hours. Thereafter,
the cells were recovered and suspended in 100 .mu.l of a buffer (5
mM Tris-HCl, pH 7.5, 2 mM MgCl.sub.2, 1 mM phenylmethylsulfonyl
fluoride), followed by sonication and centrifugation at
2000.times.g for 5 minutes. Thus, a cell extract was obtained.
[0199] (3) Assay of GnT-IV Activity
[0200] GnT-IV activity in the cell extract was determined by the
method described in Reference Example 2. The results are shown in
Table 6. Compared to the extract from cells into which pSVL vector
was introduced as a control, the extracts into which plasmid pBGT4
was introduced exhibited 44-78 times higher GnT-IV activity per
cell. From these results, it was confirmed that Gene 1 encodes
GnT-IV enzyme. Thus, GnT-IV enzyme can be produced by cultured
cells according to this method.
6 TABLE 6 Specific Activity Plasmid (pmol/hr/mg protein) Activity
Ratio pSVL 409 1 pBGT4 (#1) 29623 72 pBGT4 (#2) 31773 78 pBGT4 (#3)
20182 44 Reaction time: 4 hours Activity ratios are expressed in
relation to the total activity of pSVL that is regarded as 1.
EXAMPLE 5
Isolation and Identification of Human GnT-IVa cDNA
[0201] (1) RT-PCR
[0202] Based on the nucleotide sequence of bovine GnT-IVa obtained
in Example 3, primer h1-2F shown in SEQ ID NO: 21 and primer h1-1R
shown in SEQ ID NO: 22 were synthesized. Using total RNA from human
liver (Clontech, USA) as a template, an RT-PCR was performed with
the above primers. As a result, an amplified fragment of about 650
bp which seemed to be specific was obtained. This fragment was
subcloned, and the nucleotide sequence thereof was determined.
[0203] (2) Screening of a Library
[0204] A cDNA library from human liver (Clontech, USA) was screened
using the 685 bp DNA fragment obtained by the above RT-PCR as a
probe.
[0205] Two positive plaques of hGT4/.lambda.gt10-1 and
hGT4/.lambda.gt10-2 were obtained. The nucleotide sequences of the
inserts in these phage clones were determined. As a result,
hGT4/.lambda.gt10-1 contained a 804 bp DNA region and
hGT4/.lambda.gt10-2 contained a 2115 bp DNA region. The former
region was entirely included in the latter region. As shown in SEQ
ID NO: 23, the DNA fragment contained in hGT4/.lambda.gt10-2 had an
open reading frame (ORF) highly homologous to the amino acid
sequence of bovine GnT-IVa (96% identical). From the results
described in Example 6, it was confirmed that this ORF is human
GnT-IVa gene. The amino acid sequence of this ORF is shown in SEQ
ID NO: 24.
EXAMPLE 6
Construction of an Expression Plasmid for Human GnT-IVa Gene and a
Method for Producing Human GnT-IVa Enzyme
[0206] (1) Construction of Expression Plasmid pHGT4-1 for Human
GnT-IVa Gene
[0207] A primer (h1-7F; SEQ ID NO: 25) which introduces an XhoI
site into a region upstream of the initiation codon of human
GnT-IVa gene and another primer (h1-7R; SEQ ID NO: 26) which is
complementary to a region downstream of the termination codon of
the gene were synthesized. Using RNA from human liver (Clontech,
USA) as a template, the entire gene coding for human GnT-IVa enzyme
was amplified by RT-PCR with the above primers. The resultant
amplified fragment was inserted in the SrfI site of plasmid
pCRScript Amp SK(+) (Stratagene, DNA) in the opposite direction to
the transcription of lacZ gene. Using the resultant plasmid, it was
confirmed by nucleotide sequence analysis that the amplified
fragment encodes the amino acid sequence shown in SEQ ID NO: 24.
Further, this plasmid was digested with XhoI and SacI to obtain an
XhoI-SacI 1.7 kb fragment. This fragment was inserted bewteen the
XhoI and SacI sites of pSVL vector (Pharmacia, Sweden) to prepare
an expression plasmid pHGT4-1 for human GnT-IVa gene.
[0208] (2) Introduction of Human GnT-IVa Gene into COS7 Cells
[0209] Plasmid pHGT4-1 was introduced into COS7 cells by
electroporation. The resultant cells were cultured under 10%
CO.sub.2 at 37.degree. C. for 72 hours. Then, the cells were
harvested, suspended in 100 .mu.l of a buffer (5 mM Tris-HCl, pH
7.5, 2 mM magnesium chloride, 1 mM phenylmethylsulfon yl fluoride),
disrupted by sonication, centrifuged at 2000.times.g for 5 minutes
and collect supernatant to obtain a cell extract.
[0210] (3) Expression of Human GnT-IVa Gene in COS7 Cells
[0211] GnT-IV activity in the cell extract was determined by the
method described in Reference Example 2. The results are shown in
Table 7. Compared to the extract from cells into which pSVL vector
was introduced as a control, the extracts from cells into which
plasmid pHGT4-1 was introduced exhibited 21-28 times higher GnT-IV
activity per cell. From these results, it was confirmed that
GnT-IVa gene shown in SEQ ID NO: 23 encodes the glycosyltransferase
GnT-IV. It was also confirmed that human GnT-IVa enzyme can be
produced by cultured cells according to this method.
7 TABLE 7 Specific Activity Plasmid (pmol/hr/mg protein) Activity
Ratio pSVL 1037 1 pHGT4-1 (#1) 28951 28 pHGT4-1 (#2) 21788 21
pHGT4-2 (#1) 11024 11 pHGT4-2 (#2) 8029 8 Reaction time: 1.3 hours
Activity ratios are expressed in relation to the total activity of
pSVL that is regarded as 1.
EXAMPLE 7
Isolation and Identification of Human GnT-IVb cDNA
[0212] (1) Acquisition of Human GnT-IVa-like Gene by PCR, RT-PCR
and 5' RACE (Rapid Amplification of cDNA Ends)
[0213] Nucleotide sequences having similarity to the nucleotide
sequence of human GnT-IVa gene obtained in Example 3 were searched
for in the DNA database GenBank by BLASTN. As a result, Accession
Numbers R12057, H10557 and W16571 were found out. Then, primer
h2-45F shown in SEQ ID NO: 27 and primer h2-43R shown in SEQ ID NO:
28 were synthesized to perform a PCR using a cDNA library from
human brain of Quick Screen Human cDNA Library Panel (Clontech,
USA) as a template. The amplified fragment was subcloned into the
SrfI site of pCRScript Amp SK(+) (Stratagene, USA) and subjected to
analysis of the nucleotide sequence. Also, primer h2-2F shown in
SEQ ID NO: 29 and primer h2-1R shown in SEQ ID NO: 30 were
synthesized to perform an RT-PCR using total RNA from human lung
(Clontech, USA) as a template. As a result, an amplified fragment
of about 500 bp of the expected size was obtained. Then, this
fragment was subcloned into the SrfI site of pCRScript Amp SK(+)
(Stratagene, USA) and subjected to analysis of the nucleotide
sequence.
[0214] The thus obtained nucleotide sequences of the two DNA
fragments were overlapping with each other forming a region of 1006
bp. In this region, one reading frame which encodes the homologous
amino acid sequences to those of bovine and human GnT-IVa was
recognized. Thus, the existence of a protein relating to GnT-IVa
proteins was suggested.
[0215] Then, possible nucleotide sequences which may be an upstream
sequence to R12057 or a downstream sequence to W16571 were searched
for in the DNA database GenBank by BLASTN. As a result, R15554 was
found as an upstream sequence to R12057, and W16466 as a downstream
sequence to W16571. However, a apparently inappropriate termination
codon was contained in the ORFs deduced from these nucleotide
sequences. Therefore, in order to confirm the nucleotide sequences,
DNA fragments were obtained by RT-PCR. As primers, h2-1F shown in
SEQ ID NO: 31, h2-3F shown in SEQ ID NO: 32 and h2-8R shown in SEQ
ID NO: 33 were synthesized. With a combination of h2-1F and the
h1-1R described in Example 5, or a combination of h2-3F and h2-8R,
an RT-PCR was performed using total RNA from human liver (Clontech,
USA) as a template. Amplified fragments of about 550 bp and about
300 bp, both coinciding with the expected sizes, were detected.
Each of these fragments was subcloned in the SrfI site of pCRScript
Amp SK(+) to analyze the nucleotide sequence thereof. As a result,
it was confirmed that these fragments are respectively overlapping
with an upstream region and a downstream region to the 1006 bp
region between h2-45F and h2-1R mentioned above. In the ligated
region of 1361 bp, an ORF was found which encodes 433 amino acids
protein having high similarity to the amino acid sequences of
bovine and human GnT-IVa proteins.
[0216] However, when this ORF is compared to the amino acid
sequences of GnT-IVa proteins, it was presumed that the starting
methionine should be present in a region upstream to this ORF.
Therefore, the upstream region was obtained by 5'-RACE using Human
Lung 5'-RACE-Ready cDNA (Clontech, USA). In the first PCR, an
anchor primer and h2-5R shown in SEQ ID NO: 34 were used as
primers. In the second PCR, an anchor primer and h2-3R shown in SEQ
ID NO: 35 were used as primers. The fragments obtained by 5'-RACE
were purified, digested with EcoRI and PstI, and then separated by
agarose gel electrophoresis. A fragment of about 450 bp was
recovered from the gel. This fragment was inserted between the
EcoRI and PstI sites of pUC18 vector (Pharmacia, Sweden) to analyze
the nucleotide sequence thereof. As a result, it was confirmed that
this fragment is overlapping with a region upstream of the region
between h2-1F and h2-8R. In the ligated region of 1758 bp, one ORF
was confirmed which encodes 548 amino acids protein having high
similarity to the amino acid sequences of bovine and human GnT-IVa
proteins. The nucleotide sequence of this ORF is shown in SEQ ID
NO: 36, and the amino acid sequence thereof in SEQ ID NO: 37. From
the results described in Example 8 below, it was confirmed that
this gene is human GnT-IVb gene.
EXAMPLE 8
Construction of an Expression Plasmid for Human GnT-IVb Gene and a
Method for Producing Human GnT-IVb Enzyme
[0217] (1) Construction of Expression Plasmid pHGT4-2 for Human
GnT-IVb Gene
[0218] A primer (h2-4: SEQ ID NO: 38) which introduces an XhoI site
into a region upstream of the initiation codon of human GnT-IVb
gene, and another primer (h2-10R: SEQ ID NO: 39) which introduces
an XbaI site in a region downstream to the termination codon of the
above gene were synthesized. Using these primers, the entire ORF
coding for human GnT-IVb enzyme was amplified by RT-PCR with RNA
from human lung (Clontech, USA) as a template. The amplified
fragment was inserted into the SrfI site of plasmid pCRScript Amp
SK(+), followed by determination of the nucleotide sequence
thereof. As a result, it was confirmed that the amplified fragment
is coding for the amino acid sequence of SEQ ID NO: 37. Further,
this plasmid was digested with XhoI and XbaI to obtain an XhoI-XbaI
1.7 kb fragment. This fragment was inserted between the XhoI and
XbaI sites of pSVL vector (Pharmacia, Sweden) to construct an
expression plasmid pHGT4-2 for human GnT-IVb gene.
[0219] (2) Introduction of Human GnT-IVb Gene into COS7 Cells
[0220] Plasmid pHGT4-2 was introduced into COS7 cells by
electroporation. The resultant cells were cultured under 10%
CO.sub.2 at 37.degree. C. for 72 hours. Then, the cells were
recovered, suspended in 100 .mu.l of a buffer (5 mM Tris-HCl, pH
7.5, 2 mM magnesium chloride, 1 mM phenylmethylsulfonyl fluoride),
disrupted by sonication, centrifuged at 2000.times.g for 5 minutes
and collect supernatant to thereby obtain a cell extract.
[0221] (3) Expression of Human GnT-IVb Gene in COS7 Cells
[0222] GnT-IV activity in the cell extract was determined by the
method described in Reference Example 2. The results are shown in
Table 7 above. Compared to the extract from cells into which pSVL
vector was introduced as a control, the extracts from cells into
which plasmid pHGT4-2 was introduced exhibited 8-11 times higher
GnT-IV activity per cell. From these results, it was confirmed that
the GnT-IVb gene shown in SEQ ID NO: 36 encodes the
glycosyltransferase GnT-IV. It was also confirmed that human
GnT-IVb enzyme can be produced by cultured cells according to this
method.
EXAMPLE 9
Construction of Expression Plasmids for Bovine GnT-IVa N-Terminal
Deletion Mutants and their Expression of
[0223] (1) Construction of Expression Plasmids pSigIle93,
pSigPro113 and pSigPro142 for Bovine GnT-IVa
[0224] A primer (XhoEsig: SEQ ID NO:40) which introduces an XhoI
site into a region upstream of the signal sequence of human
erythropoietin (GenBank Accession Number X02157) and an antisense
primer (E4-1R: SEQ ID NO: 41) which ligates the C-terminus of the
above signal sequence to a part of the bovine GnT-IVa amino acid
sequence spanning from position 93 (Ile) to the end were
synthesized to amplify the signal sequence of human erythropoietin
by PCR. Also, a sense primer (E4-1F: SEQ ID NO: 42) corresponding
to the above antisense primer and a primer (4EXPR: SEQ ID NO: 20)
which introduces an XbaI site in a region downstream of the
termination codon of bovine GuT-IVa gene were synthesized to
amplify a partial sequence of bovine GnT-IVa gene by PCR. Using
portions of the resultant two PCR products as a mixed template, a
PCR was performed with primers XhoEsig and 4EXPR. The amplified
fragment was digested with XhoI and XbaI and inserted between the
XhoI and XbaI sites of pSVL vector (Pharmacia, Sweden), to thereby
construct plasmid pSigIle93 that expresses an amino acid sequence
in which the human erythropoietin signal is linked to a part of the
bovine GnT-IVa amino acid sequence spanning from position 93 to the
end.
[0225] Plasmid pSigPro113 that expresses an amino acid sequence in
which the human erythropoietin signal is linked to a part of the
bovine GnT-IVa amino acid sequence spanning from position 113 (Pro)
to the end; or plasmid pSigPro142 that expresses an amino acid
sequence in which the human erythropoietin signal is linked to a
part of the bovine GnT-IVa amino acid sequence spanning from
position 142 (Pro) to the end was constructed respectively in the
same manner as described above using E4-2R primer (SEQ ID NO: 43)
or E4-3R primer (SEQ ID NO: 44) instead of E4-1R primer; and E4-2F
primer (SEQ ID NO: 45) or E4-3F primer (SEQ ID NO: 46) instead of
E4-1F primer.
[0226] (2) Introduction of plasmids expressing Bovine GnT-IVa
N-Terminal Deletion Mutants into COS7 Cells
[0227] Plasmid pSigIle93, pSigPro113 or pSigPro142 was introduced
into COS7 cells by electroporation. The resultant cells were
cultured under 10% CO.sub.2 at 37.degree. C. for 72 hours. Then,
the cells and the culture supernatant were recovered separately.
The cells were suspended in 100 .mu.l of a buffer (5 mM Tris-HCl,
pH 7.5, 2 mM magnesium chloride, 1 mM phenylmethylsulfonyl
fluoride), disrupted by sonication and centrifuged at 2000.times.g
for 5 minutes to thereby obtain a cell extract. The culture
supernatant was concentrated to about 100 .mu.l with Centriplus 30
(Amicon).
[0228] (3) Expression of Bovine GnT-IVa N-Terminal Deletion Mutants
in COS7 Cells
[0229] GnT-IV activity in the culture supernatant and the cell
extract was determined by the method described in Reference Example
2. The results are shown in Table 8. Compared to the total activity
(i.e., activity in cells+activity in supernatant) of the cells into
which pBGT4 vector was introduced as a positive control, the total
activity of the cells into which pSigIle93 was introduced was more
than 30%. Furthermore, more than one third of the activity was
secreted into the culture supernatant. From these results, it was
found that the amino acids from the N-terminus to position 92 of
the bovine GnT-IVa amino acid sequence can be deleted while
retaining the enzyme activity. It was also shown that GnT-IVa
enzyme can be expressed secretively by using an appropriate
secretion signal.
8TABLE 8 Activity Total Ratio in Activity Activity Each Fraction
Ratio Plasmid Fraction (pmol/hr) (%) (%) pSVL Supernatant 136 0.5
1.9 pSVL Cells 384 1.4 pBGT4 Supernatant 722 2.7 100.0 pBGT4 Cells
26152 97.3 pSigIle93 Supernatant 3106 11.6 31.9 pSigIle93 Cells
5471 20.4 pSigPro113 Supernatant 312 1.2 3.4 pSigPro113 Cells 606
2.3 pSigPro142 Supernatant 219 0.8 2.2 pSigPro142 Cells 381 1.4
Reaction time: 2.5 hours The activity ratios are expressed in
percent in relation to the total activity of pBGT4 that is regarded
as 100%.
EXAMPLE 10
Construction of Expression Plasmids for Bovine GnT-IVa C-Terminal
Deletion Mutants and their Expression
[0230] (1) Construction of Expression Plasmids pCGly499, pCPro465,
pCLys432 and pCPro383 for Bovine GnT-IVa
[0231] A primer (SEQ ID NO: 19) which introduces an XhoI site into
a region upstream of the initiation codon of bovine GnT-IVa gene
and a primer (CGly499: SEQ ID NO: 47) which ligates the termination
codon after the Gly codon at position 499 and introduce an XbaI
site in a region downstream to the termination codon above were
synthesized to amplify a partial sequence of bovine GnT-IVa gene by
PCR. The amplified fragment was digested with XhoI and XbaI, and
inserted between the XhoI and XbaI sites of pSVL vector (Pharmacia,
Sweden). Thus, plasmid pCGly499 which expresses the bovine GnT-IVa
amino acid sequence up to position 499 (Glycine) was constructed.
Using CPro465 primer (SEQ ID NO: 48), CLys432 primer (SEQ ID NO:
49) or CPro383 primer (SEQ ID NO: 50) instead of CGly499 primer,
three additional plasmids were constructed in the same manner. They
were designated pCPro465 (plasmid which expresses the bovine
GnT-IVa amino acid sequence up to position 465 (Proline)); pCLys432
(plasmid which expresses the bovine GnT-IVa amino acid sequence up
to position 432 (Lysine)); and pCPro383 (plasmid which expresses
the bovine GnT-IVa amino acid sequence up to position 383
(Proline)), respectively.
[0232] (2) Introduction of plasmids expressing Bovine GnT-IVa
C-Terminal Deletion Mutants into COS7 Cells
[0233] Plasmid pCGly499, pCPro465, pCLys432 or pCPro383 was
introduced into COS7 cells by electroporation. The resultant cells
were cultured under 10% CO.sub.2 at 37.degree. C. for 72 hours.
Then, the cells were recovered and suspended in 100 .mu.l of a
buffer (5 mM Tris-HCl, pH 7.5, 2 mM magnesium chloride, 1 mM
phenylmethylsulfonyl fluoride), disrupted by sonication and
centrifuged at 2000.times.g for 5 minutes to thereby obtain a cell
extract.
[0234] (3) Expression of Bovine GnT-IVa C-Terminal Deletion Mutants
in COS7 Cells
[0235] GnT-IV activity in the cell extract was determined by the
method described in Reference Example 2. The results are shown in
Table 9. Compared to the GnT-IV activity of the extract from cells
into which pBGT4 vector was introduced as a positive control, that
activity of the extract from cells into which pCGly499, pCPro465,
pCLys432 or pCPro383 was introduced was 15.2%, 12.1%, 2.8% or
104.2% per cell, respectively. From these results, it was shown
that GnT-IV activity can be retained even if the amino acids from
position 384 to the C-terminus in the bovine GnT-IVa amino acid
sequence are deleted.
9 TABLE 9 Specific Activity Activity Ratio Plasmid (pmol/hr/mg
protein) (%) pSVL 77 1 pBGT4 14917 100 pCGly499 2263 15 pCPro465
1798 12 pCLys432 410 3 pCPro383 15551 104 Reaction Time: 2 hours
Activity ratios are expressed in percent in relation to the total
activity of pBGT4 which is regarded as 100%.
EXAMPLE 11
Construction of Plasmids to Express Various GnT-IV Genes in E. coli
and their Expression
[0236] (1) Construction of E. coli Expression Plasmid for Bovine
GnT-IVa
[0237] A primer (BSP-N: SEQ ID NO: 51) which introduces a BspHI
site in a region upstream of the initiation codon of bovine GnT-IVa
gene and another primer (C-Hd: SEQ ID NO: 52) which introduces a
HindIII site in a region downstream of the termination codon were
synthesized to amplify the entire open reading frame of bovine
GnT-IVa gene by PCR. The amplified fragment was digested with BspHI
and HindIII, and introduced between the NcoI and HindIII sites of
pTrc99A vector (Pharmacia, Sweden) to thereby construct plasmid
pEBGT4. Using BSP-sN primer (SEQ ID NO: 53) instead of BSP-N primer
together with C-Hd primer, plasmid pEIle93 was constructed in a
similar manner. Further, a gene coding for the open reading frame
in which His-Tag is added to the C-terminus was amplified using
BSP-N primer, a primer (CH-Hd: SEQ ID NO: 54) which can introduce
His-Tag, a termination codon and a HindIII site in a region
downstream of the C-terminus of bovine GnT-IVa gene and a primer
(H-Hd: SEQ ID NO: 55) which has His-Tag, a termination codon and a
HindIII site, to thereby construct plasmid pEBGT4+His in a similar
manner.
[0238] (2) Construction of E. coli Expression Plasmid for Human
GnT-IVa Gene and Human GnT-IVb Gene
[0239] A primer (4aBSPIL94: SEQ ID NO: 56) which introduces an
initiation codon and an Ile codon in a region upstream of position
94 (Leu) of the human GnT-IVa amino acid sequence and which can
also introduce a BspHI site at a region further upstream thereof; a
primer (4aCH-Hd: SEQ ID NO: 57) which introduces His-Tag, a
termination codon and a HindIII site in a region downstream of the
C-terminal amino acid; and H-Hd primer were synthesized to amplify
a gene fragment composed a partial sequence of the human GnT-IVa
amino acid sequence to which a sequence encoding His-Tag is added.
The amplified fragment was digested with BspHI and HindIII, and
inserted between the NcoI and HindIII sites of pTrc99A vector
(Pharmacia, Sweden) to thereby construct plasmid pMA4a+His.
Further, using CP383H-Hd primer (SEQ ID NO: 58) instead of 4aCH-Hd
primer, plasmid pCQre+His was constructed in a similar manner (FIG.
18). A fragment of human GnT-IVb gene was amplified using a primer
(4bBSP-N: SEQ ID NO: 59) which introduces a BspHI site in a region
upstream of the initiation codon of human GnT-IVb gene and 4bSACR
primer (SEQ ID No: 60), digested with BspHI and SacI, and then
inserted between the NcoI and SacI sites of pTrc99A vector
(Pharmacia, Sweden). Between the SacI and HindIII sites of the
resultant plasmid, a partial length of human GnT-IVb gene amplified
using 4bSACF primer (SEQ ID NO: 61), a primer (4bCH-Hd: SEQ ID NO:
62) which introduces His-Tag at the C-terminus of the human GnT-IVb
amino acid sequence and H-Hd primer, and digested with Sac I and
HindIII was inserted to thereby achieve plasmid pEHGT4-2+His.
Further, a partial sequence of human GnT-IVb gene was amplified
using a primer (4bNCOG91: SEQ ID NO: 63) which introduces an NcoI
site and an initiation codon in a region upstream of position 91
(Gly) of the human GnT-IVb amino acid sequence, 4bCH-Hd primer and
H-Hd primer, digested with NcoI and HindIII, and then inserted
between the NcoI and HindIII sites of pTrc99A vector (Pharmacia,
Sweden) to thereby construct plasmid pMA4b+His.
[0240] (3) Introduction of Each Expression Plasmid into E. coli
BL21 Strain
[0241] Each expression plasmid was introduced into competent cells
of E. coli BL21 strain prepared by the calcium method. The
resultant cells were cultured on LB agar plate containing 100
.mu.g/ml ampicillin. The resultant colonies of E. coli transformed
with each plasmid were inoculated into LB liquid medium and
cultured under shaking at 37.degree. C. overnight. Then, the
culture was inoculated into a fresh LB liquid medium to give a
concentration of 2%. While the turbidity (OD 595 nm) of the culture
fluid was about 0.1 to 0.2, IPTG (isopropyl
b-D-thiogalactopyranoside) was added thereto to give a final
concentration of 1 mM. The cells were cultured at 37.degree. C. for
2 hours or at 25.degree. C. for 4 hours. Then, 500 .mu.l of the
cells was harvested. The cell pellet was suspended in 50 .mu.l of a
buffer (5 mM Tris-HCl, pH 7.5, 2 mM MgCl.sub.2, 1 mM
phenylmethylsulfonyl fluoride), disrupted by sonication and
centrifuged at 2000.times.g for 5 minutes to obtain a cell extract
as a supernatant.
[0242] (4) Expression of Each Expression Plasmid in E. coli BL21
Strain
[0243] GnT-IV activity in the cell extract was determined by the
method described in Reference Example 2. Table 10 shows the results
of the expression of the bovine gene. Although the extract from E.
coli cells into which pTrc99A vector was introduced as a control
had little GnT-IV, the extract from E. coli cells into which pEBGT4
was introduced had definite GnT-IV activity. From these results, it
was demonstrated that GnT-IV enzyme can be produced by E. coli. The
His-tag sequence added to the C-terminus of bovine GnT-IVa did not
influence greatly upon GnT-IV activity. Thus, it was shown that an
appropriate tag sequence can be added to GnT-IV enzyme. The mutant
(pEIle93) in which the N-terminal 92 amino acid are deleted
exhibited stronger GnT-IV activity. Thus, the expression of
variants of GnT-IV enzyme which was confirmed in animal cells was
also shown possible in E. coli.
10 TABLE 10 Activity Activity Ratio Plasmid (pmol/hr/mg protein)
(%) pTrc99A 0 0 pEBGT4 4611 100 pEBGT4 + His 3090 67 pEIle93 5841
127 Reaction time: 3.0 hours After IPTG addition, cells were
cultured at 37.degree. C. for 2 hours. Activity ratios are
expressed in percent in relation to the total activity of pEBGT4
which is regarded as 100%.
[0244] Table 11 shows the results of the expression of the human
gene. Compared to the extract from E. coli cells into which pTrc99A
vector was introduced as a control, the extract from E. coli cells
into which any of the expression plasmids was introduced had GnT-IV
activity significantly. As shown in bovine GnT-IVa enzyme, it was
also possible in human GnT-IVa and GnT-IVb enzymes to delete an
N-terminal sequence while retaining the activity. Further, the
human GnT-IVa enzyme which has both N-terminal deletion and
C-terminal deletion exhibited high GnT-IV activity (pCore+His).
This shows that those portions deleted in this mutant are not
essential for GnT-IV activity.
11 TABLE 11 Activity Activity Ratio Plasmid (pmol/hr/mg protein)
(%) pTrc99A 0 0 pEBGT4 + His 21390 637 pMA4a + His 3359 100 pCore +
His 39766 1184 pEHGT4-2 + His 270 8 pMA4b + His 2812 84 Reaction
time: 4.0 hours After IPTG addition, cells were cultured at
25.degree. C. for 4 hours. Activity ratios are expressed in percent
in relation to the total activity of pMA4a + His which is regarded
as 100%.
EXAMPLE 12
Conversion of the Sugar Chain Branching Structure of a Recombinant
Erythropoietin (EPO) by Introducing Bovine or Human GnT-IVa Gene
into EPO-Producing CHO Cells
[0245] (1) Introduction of GnT-IV Expression Plasmid into
EPO-Producing CHO Cells
[0246] EPO-producing CHO cell clones were created according to the
method disclosed in Japanese Examined Patent Publication No.
2-17156.
[0247] GnT-IVa expression plasmid pBGT4 or pHGT4-1 was introduced
into the resultant cell clones MO1 and H-5 by electroporation. In
the introduction, 15 .mu.g of the expression plasmid and 1.5 .mu.g
of a drug resistance marker plasmid (pSV2bsr from Kaken
Pharmaceutical or pMAMneo from Clontech) were used in mixture. The
electroporated cells were cultured under 10% CO.sub.2 at 37.degree.
C. for about 60 hours. Then, blasticidin S (Kaken Pharmaceuticals)
(final concentration: 10 .mu.g/ml) or geneticin (Life Technologies,
Inc.) (final concentration: 500 .mu.g/ml) was added to the medium,
in which the cells were cultured for another 10 days to 2 weeks.
Thus, clones resistant to either of two drugs were isolated.
[0248] (2) Confirmation of the Expression of the Introduced GnT-IV
Genes in EPO-Producing CHO Cell Clones
[0249] EPO-producing CHO cell clones (initial clones) and
individual drug resistant clones were cultured in an appropriate
scale. Total RNA from each clone was purified. Then, RNA dot blot
analysis was performed using a part of GnT-IVa gene as a probe to
thereby examine the amount of GnT-IVa mRNA. Further, GnT-IV
activity expressed in the initial clones and the drug resistant
clones was determined by the assay described in Reference Example
2. Those clones which gave a strong signal in RNA dot blot analysis
and yet exhibited higher GnT-IV activity than the initial clones
were selected and used for EPO production. The selected clones had
increased GnT-IV activity; for example, MO1(bovine GnT-IV)#36
exhibited about 104-fold increase over MO1 clone, and H-5(human
GnT-IV)#23 exhibited about 125-fold increase over H-5 clone.
[0250] (3) Production of EPO using GnT-IV Gene-Introduced
EPO-Producing CHO Cell Clones
[0251] EPO is expressed secretively into culture fluid. Then,
EPO-producing CHO cell clones MO1 and H-5, and the above-mentioned
clones MO1(bovine GnT-IV)#36 and H-5(human GnT-IV)#23 were cultured
in roller bottles. First, each clone was adhesion-cultured in a
growth medium, and then 1.5.times.10.sup.7 cells were transferred
to a 850 cm.sup.2 roller bottle containing 200 ml of a growth
medium. The cells were cultured under 10% CO.sub.2 at 37.degree. C.
for 3 days so that they adhered to the bottle uniformly.
Thereafter, the growth medium was removed, and the cells were
washed with PBS buffer. Then, 200 ml of a serum-free medium was
added to the bottle, in which the cells were cultured under 10%
CO.sub.2 at 37.degree. C. for 7 days. Thereafter, the culture
supernatant was recovered.
[0252] As a growth medium, D-MEM/F12 mixed medium supplemented with
5% fetal bovine serum, 290 mg/liter L-glutamic acid, 1.times.MEM
non-essential amino acid solution and 100 nM methotrexate was used.
As a serum-free medium, the above medium without fetal bovine serum
was used. EPO contained in each of the serum-free culture
supernatants was quantitatively determined by ELISA using
anti-human EPO antibody.
[0253] (4) Analysis of EPOs Produced by GnT-IV Gene-Introduced or
-Non-Introduced Clones Based on Their Sugar Chain Structures
[0254] A recombinant EPO does not exist as a single molecule on
isoelectric focusing gel; it is mixture of molecules with various
electric charges. Since the protein moiety does not vary, it has
been shown that the difference in electric charge among these
molecules is based on the difference in sugar chain structure; such
mixture of molecules is called glycoforms [Watson, E. and Yao, F.,
Anal. Biochem. (1993), 210, 389-93]. EPO has three Asn-linked sugar
chains; the branching structures of individual sugar chains vary
from biantennary to tetraantennary. Gal (galactose) is further
attaching to the end of each of branched GlcNAc's, and sialic acid
is further attaching to this Gal. Therefore, if the degree of sugar
chain branching is increased by the introduction of GnT-IV gene,
the number of sialic acid molecules attaching to Gal should
increase and, thus, the content of glycoforms with low isoelectric
point should increase. Then, the inventors performed analysis by
isoelectric focusing to detect changes in the sugar chain structure
of the EPOs produced by GnT-IV gene-introduced EPO-producing
cells.
[0255] For the isoelectric focusing, Multiphor II equipment
manufactured by Pharmacia was used. The gel was composed of 5%
acrylamide (30:0.8) and 1.5% Pharmalyte 2.5-5 (Pharmacia). As the
(+) electrode solution, 0.1 M sulfuric acid was used. As the (-)
electrode solution, 0.2 M L-histidine was used. After the
isoelectric focusing, samples were electrophoretically transferred
onto a PVDF membrane, followed by Western blot analysis using
anti-EPO mouse monoclonal antibody to detect the bands of
individual glycoforms of the EPOs. Briefly, the serum-free culture
supernatant of each cell clone was concentrated to about 7 to
1000-fold with Centriplus 30 and Microcon 30 (both manufactured by
Amicon), if necessary. At the beginning, about 50-100 IU of EPO was
used as a sample, but this amount was adjusted appropriately so
that the intensities of bands detected by Western blot analysis
would be almost equal between samples.
[0256] When the EPO from MO1 clone was compared to the EPO from
MO1(bovine GnT-IV)#36 clone, it was confirmed that the positions of
major glycoforms in the latter show a shift to the low pI side
(+electrode solution side) by at least one glycoform (FIG. 19).
From this result, it is thought that the GnT-IV enzyme expressed as
a result of the gene introduction increased the number of GlcNAc
branches in the Asn-linked sugar chains attaching to EPO, thus
increasing the number of sialic acid molecules attaching to
increase the content of glycoforms with low isoelectric points. A
similar analysis was performed on H-5 clone and H-5(human
GnT-IV)#23 clone. As a result, it was also found that the positions
of major EPO glycoforms in the latter shift to the low pI side
(FIG. 19).
[0257] From the above, it was demonstrated that it is possible to
modify the structure of Ans-linked sugar chains of the protein
produced by the cell by introducing a GnT-IV gene into any
cell.
INDUSTRIAL APPLICABILITY
[0258] According to the present invention, a novel .beta.1.fwdarw.4
N-acetylglucosaminyltransferase (GnT-IV), a method for producing
the GnT-IV enzyme and a gene coding for the GnT-IV are provided.
With the GnT-IV of the present invention, it has become possible to
produce a glycocoujugate having a branching structure which could
not be formed with conventional glycosyltransferases. Thus, the
GnT-IV of the invention is useful not only for producing or
improving glycoconjugate type pharmaceuticals, reagents and foods,
but also for modifying the sugar chain structure of any
biopolymer.
[0259] The GnT-IV gene of the invention is also useful for
diagnosing or treating diseases such as cancer and for modifying
the sugar chain structure of glycoconjugate products produced by
microorganisms.
[0260] Further, an antibody or anti-serum raised against the GnT-IV
protein of the invention as an antigen, or a part or all of the
GnT-IV gene of the invention as a probe is useful for
characterizing microorganisms, cultured cells, various animal
tissues, blood cells and blood or for diagnosing diseased cells or
tissues such as cancer.
Sequence CWU 1
1
63 1 8 PRT Bovine 1 Asp Asn Leu Tyr Pro Glu Glu Lys 1 5 2 11 PRT
Bovine 2 Asp Tyr Val Asn Gly Val Val Ala Asn Glu Lys 1 5 10 3 21
PRT Bovine 3 Glu Ile Ser Ser Gly Leu Val Glu Ile Ile Ser Pro Pro
Glu Ser Tyr 1 5 10 15 Tyr Pro Asp Leu Thr 20 4 8 PRT Bovine 4 Glu
Arg Val Arg Trp Arg Thr Lys 1 5 5 15 PRT Bovine 5 Lys Gln Asn Leu
Asp Tyr Cys Phe Leu Met Met Tyr Ala Gln Glu 1 5 10 15 6 6 PRT
Bovine 6 Asp His Ile Leu Trp Val 1 5 7 14 PRT Bovine 7 Lys Ile His
Val Asn Pro Pro Ala Glu Val Ser Thr Ser Leu 1 5 10 8 10 PRT Bovine
8 Lys Val Tyr Gln Gly His Thr Leu Glu Lys 1 5 10 9 10 PRT Bovine 9
Asp Phe Phe Trp Ala Ile Thr Pro Val Ala 1 5 10 10 6 PRT Bovine 10
Asp Tyr Ile Leu Phe Lys 1 5 11 15 PRT Bovine 11 Asp Lys Pro Val Asn
Val Glu Ser Tyr Leu Phe His Ser Gly Asn 1 5 10 15 12 10 PRT Bovine
12 Asp Ile Leu Leu Asn Thr Thr Val Glu Val 1 5 10 13 9 PRT Bovine
13 Lys Ser Glu Gly Leu Asp Ile Ser Lys 1 5 14 8 PRT Bovine 14 Asp
Gly Tyr Phe Arg Ile Gly Lys 1 5 15 29 DNA Artificial Sequence
Description of Artificial Sequence Primer 15 aaratycayg tbaaycchcc
hgcngargt 29 16 35 DNA Artificial Sequence Description of
Artificial Sequence Primer 16 tgraavarrt arswytcvac rttvacdggy
ttrtc 35 17 2246 DNA Bovine CDS (288)..(1892) 17 ggcggctgct
cggtggcggc tcgtcggcgg ccgcggcagg actggcagcg ccggcggcgg 60
ggagaaagaa gcatccacct atgaagaccg tgcagacagt cctgaataat aattgtgaat
120 ggtgtggctg ccagactagt tctgctgagc atctgaaatg aacctctcct
attgattgtt 180 tcagttggcc ccgagccagg agtactgggt ttgcttgact
tcaggataaa aagaaacgga 240 cttggttatc atcgtaaaca tatgaaccag
tgtgatggtg aaatgag atg agg ctc 296 Met Arg Leu 1 cga aat gga act
gta gcc act gtt tta gca ttt atc acc tcg ttc ctc 344 Arg Asn Gly Thr
Val Ala Thr Val Leu Ala Phe Ile Thr Ser Phe Leu 5 10 15 act tta tct
tgg tat aca aca tgg caa aat ggg aaa gaa aaa gtg att 392 Thr Leu Ser
Trp Tyr Thr Thr Trp Gln Asn Gly Lys Glu Lys Val Ile 20 25 30 35 gct
tat caa cga gaa ttt ctt gct ctg aaa gaa cgt ctc cga ata gct 440 Ala
Tyr Gln Arg Glu Phe Leu Ala Leu Lys Glu Arg Leu Arg Ile Ala 40 45
50 gaa cat cga atc tct cag cgc tct tct gag ctc agt gcc att gta cag
488 Glu His Arg Ile Ser Gln Arg Ser Ser Glu Leu Ser Ala Ile Val Gln
55 60 65 caa ttc aag cgt gta gaa gca gaa aca aac agg agt aag gat
cca gtg 536 Gln Phe Lys Arg Val Glu Ala Glu Thr Asn Arg Ser Lys Asp
Pro Val 70 75 80 aat aaa ttt tca gat gat acc cta aag ata cta aag
gag tta aca agc 584 Asn Lys Phe Ser Asp Asp Thr Leu Lys Ile Leu Lys
Glu Leu Thr Ser 85 90 95 aaa aag tct ctt caa gtg cca agt att tat
tat cat ttg cct cat tta 632 Lys Lys Ser Leu Gln Val Pro Ser Ile Tyr
Tyr His Leu Pro His Leu 100 105 110 115 ttg caa aat gaa gga agc ctt
caa cct gcc gtg cag atc gga aat gga 680 Leu Gln Asn Glu Gly Ser Leu
Gln Pro Ala Val Gln Ile Gly Asn Gly 120 125 130 cga aca gga gtt tca
ata gta atg gga att cct aca gtg aag aga gaa 728 Arg Thr Gly Val Ser
Ile Val Met Gly Ile Pro Thr Val Lys Arg Glu 135 140 145 gtt aaa tct
tac ctc ata gaa act ctt cat tcc ctt att gat aat ctg 776 Val Lys Ser
Tyr Leu Ile Glu Thr Leu His Ser Leu Ile Asp Asn Leu 150 155 160 tat
cct gaa gag aag ttg gac tgt gtt ata gta gtc ttc ata gga gag 824 Tyr
Pro Glu Glu Lys Leu Asp Cys Val Ile Val Val Phe Ile Gly Glu 165 170
175 aca gat act gat tat gta aat ggt gtt gta gcc aac ctg gag aaa gaa
872 Thr Asp Thr Asp Tyr Val Asn Gly Val Val Ala Asn Leu Glu Lys Glu
180 185 190 195 ttt tct aaa gaa atc agt tct ggc ttg gtg gaa ata ata
tca cct cct 920 Phe Ser Lys Glu Ile Ser Ser Gly Leu Val Glu Ile Ile
Ser Pro Pro 200 205 210 gaa agc tat tat cct gac ctg acg aac tta aag
gag aca ttt gga gat 968 Glu Ser Tyr Tyr Pro Asp Leu Thr Asn Leu Lys
Glu Thr Phe Gly Asp 215 220 225 tct aaa gaa aga gta aga tgg aga aca
aag caa aac cta gat tat tgt 1016 Ser Lys Glu Arg Val Arg Trp Arg
Thr Lys Gln Asn Leu Asp Tyr Cys 230 235 240 ttt cta atg atg tat gct
cag gaa aaa ggc aca tac tac atc cag ctt 1064 Phe Leu Met Met Tyr
Ala Gln Glu Lys Gly Thr Tyr Tyr Ile Gln Leu 245 250 255 gaa gat gat
att att gtc aaa cag aat tac ttt aac acc ata aag aat 1112 Glu Asp
Asp Ile Ile Val Lys Gln Asn Tyr Phe Asn Thr Ile Lys Asn 260 265 270
275 ttt gca ctt caa ctt tct tct gag gaa tgg atg ata ctt gag ttc tcc
1160 Phe Ala Leu Gln Leu Ser Ser Glu Glu Trp Met Ile Leu Glu Phe
Ser 280 285 290 cag ctg gga ttc att ggt aaa atg ttt caa gca cct gac
cca ctc ctg 1208 Gln Leu Gly Phe Ile Gly Lys Met Phe Gln Ala Pro
Asp Pro Leu Leu 295 300 305 att gtg gaa ttc ata ttt atg ttc tat aag
gag aag ccc atc gac tgg 1256 Ile Val Glu Phe Ile Phe Met Phe Tyr
Lys Glu Lys Pro Ile Asp Trp 310 315 320 ctc ttg gac cat att ctg tgg
gtc aaa gtc tgc aac ccg gaa aaa gat 1304 Leu Leu Asp His Ile Leu
Trp Val Lys Val Cys Asn Pro Glu Lys Asp 325 330 335 gca aaa cac tgt
gat cga cag aag gca aat ctg cga att cgt ttc aga 1352 Ala Lys His
Cys Asp Arg Gln Lys Ala Asn Leu Arg Ile Arg Phe Arg 340 345 350 355
ccg tcc ctt ttc caa cac gtt ggt ctg cat tct tca ctc aca gga aaa
1400 Pro Ser Leu Phe Gln His Val Gly Leu His Ser Ser Leu Thr Gly
Lys 360 365 370 att cag aaa ctc acg gat aaa gat tac atg aaa cca tta
ctg ctc aaa 1448 Ile Gln Lys Leu Thr Asp Lys Asp Tyr Met Lys Pro
Leu Leu Leu Lys 375 380 385 atc cat gta aac ccc cct gca gag gta tct
act tct ttg aag gtc tac 1496 Ile His Val Asn Pro Pro Ala Glu Val
Ser Thr Ser Leu Lys Val Tyr 390 395 400 caa ggt cat aca ctg gag aaa
act tac atg ggt gag gac ttc ttc tgg 1544 Gln Gly His Thr Leu Glu
Lys Thr Tyr Met Gly Glu Asp Phe Phe Trp 405 410 415 gct ata acc cca
gta gct gga gac tac atc cta ttt aaa ttc gac aag 1592 Ala Ile Thr
Pro Val Ala Gly Asp Tyr Ile Leu Phe Lys Phe Asp Lys 420 425 430 435
cca gtc aat gtg gaa agt tat ttg ttc cat agt ggc aac cag gat cat
1640 Pro Val Asn Val Glu Ser Tyr Leu Phe His Ser Gly Asn Gln Asp
His 440 445 450 cca ggg gat att ctg ctc aac aca acg gtg gaa gtt ctg
cct ttg aag 1688 Pro Gly Asp Ile Leu Leu Asn Thr Thr Val Glu Val
Leu Pro Leu Lys 455 460 465 agt gaa ggt ttg gac atc agc aaa gaa acc
aaa gac aaa cga tta gaa 1736 Ser Glu Gly Leu Asp Ile Ser Lys Glu
Thr Lys Asp Lys Arg Leu Glu 470 475 480 gat ggc tat ttc aga ata ggg
aaa ttt gaa aac ggt gtt gcg gaa ggg 1784 Asp Gly Tyr Phe Arg Ile
Gly Lys Phe Glu Asn Gly Val Ala Glu Gly 485 490 495 atg gtg gat ccc
agc cta aac ccc att tcg gcc ttc cga ctt tca gtt 1832 Met Val Asp
Pro Ser Leu Asn Pro Ile Ser Ala Phe Arg Leu Ser Val 500 505 510 515
att cag aat tct gct gtt tgg gcc att ctt aat gag atc cat att aaa
1880 Ile Gln Asn Ser Ala Val Trp Ala Ile Leu Asn Glu Ile His Ile
Lys 520 525 530 aaa gtc aca aac tgaccatctc tactaagaaa ccaacacatt
ttttccctgt 1932 Lys Val Thr Asn 535 gaatttgttg attaaagaca
gctgagcacg tacctttttt tggtaacttg aattctacct 1992 ctcgcgaaat
ctactgtaga taaaatgatt gtcatatttc cacttggaaa atgaatctcc 2052
cacggataat tgtattcatt tgaatctaag ctgtcctcca gttttaactc aactcaaacg
2112 ttttacagtt atgacagcct gttaatatga cttgtactat tttggtatta
tactaataca 2172 taagagttgt acatattgtt acattcatta aatttgagaa
aaattaatgt taaatacatt 2232 ttatgaacgg gccg 2246 18 535 PRT Bovine
18 Met Arg Leu Arg Asn Gly Thr Val Ala Thr Val Leu Ala Phe Ile Thr
1 5 10 15 Ser Phe Leu Thr Leu Ser Trp Tyr Thr Thr Trp Gln Asn Gly
Lys Glu 20 25 30 Lys Val Ile Ala Tyr Gln Arg Glu Phe Leu Ala Leu
Lys Glu Arg Leu 35 40 45 Arg Ile Ala Glu His Arg Ile Ser Gln Arg
Ser Ser Glu Leu Ser Ala 50 55 60 Ile Val Gln Gln Phe Lys Arg Val
Glu Ala Glu Thr Asn Arg Ser Lys 65 70 75 80 Asp Pro Val Asn Lys Phe
Ser Asp Asp Thr Leu Lys Ile Leu Lys Glu 85 90 95 Leu Thr Ser Lys
Lys Ser Leu Gln Val Pro Ser Ile Tyr Tyr His Leu 100 105 110 Pro His
Leu Leu Gln Asn Glu Gly Ser Leu Gln Pro Ala Val Gln Ile 115 120 125
Gly Asn Gly Arg Thr Gly Val Ser Ile Val Met Gly Ile Pro Thr Val 130
135 140 Lys Arg Glu Val Lys Ser Tyr Leu Ile Glu Thr Leu His Ser Leu
Ile 145 150 155 160 Asp Asn Leu Tyr Pro Glu Glu Lys Leu Asp Cys Val
Ile Val Val Phe 165 170 175 Ile Gly Glu Thr Asp Thr Asp Tyr Val Asn
Gly Val Val Ala Asn Leu 180 185 190 Glu Lys Glu Phe Ser Lys Glu Ile
Ser Ser Gly Leu Val Glu Ile Ile 195 200 205 Ser Pro Pro Glu Ser Tyr
Tyr Pro Asp Leu Thr Asn Leu Lys Glu Thr 210 215 220 Phe Gly Asp Ser
Lys Glu Arg Val Arg Trp Arg Thr Lys Gln Asn Leu 225 230 235 240 Asp
Tyr Cys Phe Leu Met Met Tyr Ala Gln Glu Lys Gly Thr Tyr Tyr 245 250
255 Ile Gln Leu Glu Asp Asp Ile Ile Val Lys Gln Asn Tyr Phe Asn Thr
260 265 270 Ile Lys Asn Phe Ala Leu Gln Leu Ser Ser Glu Glu Trp Met
Ile Leu 275 280 285 Glu Phe Ser Gln Leu Gly Phe Ile Gly Lys Met Phe
Gln Ala Pro Asp 290 295 300 Pro Leu Leu Ile Val Glu Phe Ile Phe Met
Phe Tyr Lys Glu Lys Pro 305 310 315 320 Ile Asp Trp Leu Leu Asp His
Ile Leu Trp Val Lys Val Cys Asn Pro 325 330 335 Glu Lys Asp Ala Lys
His Cys Asp Arg Gln Lys Ala Asn Leu Arg Ile 340 345 350 Arg Phe Arg
Pro Ser Leu Phe Gln His Val Gly Leu His Ser Ser Leu 355 360 365 Thr
Gly Lys Ile Gln Lys Leu Thr Asp Lys Asp Tyr Met Lys Pro Leu 370 375
380 Leu Leu Lys Ile His Val Asn Pro Pro Ala Glu Val Ser Thr Ser Leu
385 390 395 400 Lys Val Tyr Gln Gly His Thr Leu Glu Lys Thr Tyr Met
Gly Glu Asp 405 410 415 Phe Phe Trp Ala Ile Thr Pro Val Ala Gly Asp
Tyr Ile Leu Phe Lys 420 425 430 Phe Asp Lys Pro Val Asn Val Glu Ser
Tyr Leu Phe His Ser Gly Asn 435 440 445 Gln Asp His Pro Gly Asp Ile
Leu Leu Asn Thr Thr Val Glu Val Leu 450 455 460 Pro Leu Lys Ser Glu
Gly Leu Asp Ile Ser Lys Glu Thr Lys Asp Lys 465 470 475 480 Arg Leu
Glu Asp Gly Tyr Phe Arg Ile Gly Lys Phe Glu Asn Gly Val 485 490 495
Ala Glu Gly Met Val Asp Pro Ser Leu Asn Pro Ile Ser Ala Phe Arg 500
505 510 Leu Ser Val Ile Gln Asn Ser Ala Val Trp Ala Ile Leu Asn Glu
Ile 515 520 525 His Ile Lys Lys Val Thr Asn 530 535 19 31 DNA
Artificial Sequence Description of Artificial Sequence Primer 19
ccctcgagat gaggctccga aatggaactg t 31 20 31 DNA Artificial Sequence
Description of Artificial Sequence Primer 20 tttctagatc agtttgtgac
ttttttaata t 31 21 24 DNA Artificial Sequence Description of
Artificial Sequence Primer 21 acgattgtgc aacagttcaa gcgt 24 22 24
DNA Artificial Sequence Description of Artificial Sequence Primer
22 gggagaactc caggatcatc cagt 24 23 2115 DNA Homo sapiens CDS
(136)..(1740) 23 gaaatgaacc tctcttattg atttttattg gcctagagcc
aggagtactg cattcagttg 60 actttcaggg taaaaagaaa acagtcctgg
ttgttgtcat cataaacata tggaccagtg 120 tgatggtgaa atgag atg agg ctc
cgc aat gga act gta gcc act gct tta 171 Met Arg Leu Arg Asn Gly Thr
Val Ala Thr Ala Leu 1 5 10 gca ttt atc act tcc ttc ctt act ttg tct
tgg tat act aca tgg caa 219 Ala Phe Ile Thr Ser Phe Leu Thr Leu Ser
Trp Tyr Thr Thr Trp Gln 15 20 25 aat ggg aaa gaa aaa ctg att gct
tat caa cga gaa ttc ctt gct ttg 267 Asn Gly Lys Glu Lys Leu Ile Ala
Tyr Gln Arg Glu Phe Leu Ala Leu 30 35 40 aaa gaa cgt ctt cga ata
gct gaa cac aga atc tca cag cgc tct tct 315 Lys Glu Arg Leu Arg Ile
Ala Glu His Arg Ile Ser Gln Arg Ser Ser 45 50 55 60 gaa tta aat acg
att gtg caa cag ttc aag cgt gta gga gca gaa aca 363 Glu Leu Asn Thr
Ile Val Gln Gln Phe Lys Arg Val Gly Ala Glu Thr 65 70 75 aat gga
agt aag gat gcg ttg aat aag ttt tca gat aat acc cta aag 411 Asn Gly
Ser Lys Asp Ala Leu Asn Lys Phe Ser Asp Asn Thr Leu Lys 80 85 90
ctg tta aag gag tta aca agc aaa aaa tct ctt caa gtg cca agt att 459
Leu Leu Lys Glu Leu Thr Ser Lys Lys Ser Leu Gln Val Pro Ser Ile 95
100 105 tat tat cat ttg cct cat tta ttg aaa aat gaa gga agt ctt caa
cct 507 Tyr Tyr His Leu Pro His Leu Leu Lys Asn Glu Gly Ser Leu Gln
Pro 110 115 120 gct gta cag att ggc aac gga aga aca gga gtt tca ata
gtc atg ggc 555 Ala Val Gln Ile Gly Asn Gly Arg Thr Gly Val Ser Ile
Val Met Gly 125 130 135 140 att ccc aca gtg aag aga gaa gtt aaa tct
tac ctc ata gaa act ctt 603 Ile Pro Thr Val Lys Arg Glu Val Lys Ser
Tyr Leu Ile Glu Thr Leu 145 150 155 cat tcc ctt att gat aac ctg tat
cct gaa gag aag ttg gac tgt gtt 651 His Ser Leu Ile Asp Asn Leu Tyr
Pro Glu Glu Lys Leu Asp Cys Val 160 165 170 ata gta gtc ttc ata gga
gag aca gat att gat tat gta cat ggt gtt 699 Ile Val Val Phe Ile Gly
Glu Thr Asp Ile Asp Tyr Val His Gly Val 175 180 185 gta gcc aac ctg
gag aaa gaa ttt tct aaa gaa atc agt tct ggc ttg 747 Val Ala Asn Leu
Glu Lys Glu Phe Ser Lys Glu Ile Ser Ser Gly Leu 190 195 200 gtg gaa
gtc ata tca ccc cct gaa agc tat tat cct gac ttg aca aac 795 Val Glu
Val Ile Ser Pro Pro Glu Ser Tyr Tyr Pro Asp Leu Thr Asn 205 210 215
220 cta aag gag aca ttt gga gac tcc aaa gaa aga gta aga tgg aga aca
843 Leu Lys Glu Thr Phe Gly Asp Ser Lys Glu Arg Val Arg Trp Arg Thr
225 230 235 aag caa aac cta gat tac tgt ttt cta atg atg tat gct caa
gaa aag 891 Lys Gln Asn Leu Asp Tyr Cys Phe Leu Met Met Tyr Ala Gln
Glu Lys 240 245 250 ggc ata tat tac att cag ctt gaa gat gat att att
gtc aaa caa aat 939 Gly Ile Tyr Tyr Ile Gln Leu Glu Asp Asp Ile Ile
Val Lys Gln Asn 255 260 265 tat ttt aat acc ata aaa aat ttt gca ctt
caa ctt tct tct gag gaa 987 Tyr Phe Asn Thr Ile Lys Asn Phe Ala Leu
Gln Leu Ser Ser Glu Glu 270 275 280 tgg atg att cta gag ttt tcc cag
ctg ggc ttc att ggt aaa atg ttt 1035 Trp Met Ile Leu Glu Phe Ser
Gln Leu Gly Phe Ile Gly Lys Met Phe 285 290 295 300 caa gcg ccg gat
ctt act ctg att gta gaa ttc ata ttc atg ttt tac 1083 Gln Ala Pro
Asp Leu Thr Leu Ile Val Glu Phe Ile Phe Met Phe Tyr 305 310 315 aag
gag aaa ccc att gat tgg ctc ctg gac cat att ctc tgg gtg aaa 1131
Lys Glu Lys Pro Ile Asp Trp Leu Leu Asp His Ile Leu Trp Val Lys 320
325 330 gtc tgc aac cct gaa aaa gat gca aaa cat tgt gat aga cag aaa
gca 1179 Val Cys Asn Pro Glu Lys Asp Ala Lys His Cys Asp Arg Gln
Lys Ala 335 340 345 aat ctg cga att cgc ttc aga cct tcc ctt ttc caa
cat gtt ggt ctg 1227 Asn Leu Arg Ile Arg Phe Arg
Pro Ser Leu Phe Gln His Val Gly Leu 350 355 360 cac tca tca cta tca
gga aaa atc caa aaa ctc acg gat aaa gat tat 1275 His Ser Ser Leu
Ser Gly Lys Ile Gln Lys Leu Thr Asp Lys Asp Tyr 365 370 375 380 atg
aaa cca tta ctt ctt aaa atc cat gta aac cca cct gcg gag gta 1323
Met Lys Pro Leu Leu Leu Lys Ile His Val Asn Pro Pro Ala Glu Val 385
390 395 tct act tcc ttg aag gtc tac caa ggg cat acg ctg gag aaa act
tac 1371 Ser Thr Ser Leu Lys Val Tyr Gln Gly His Thr Leu Glu Lys
Thr Tyr 400 405 410 atg gga gag gat ttc ttc tgg gct atc aca ccg ata
gct gga gac tac 1419 Met Gly Glu Asp Phe Phe Trp Ala Ile Thr Pro
Ile Ala Gly Asp Tyr 415 420 425 atc ttg ttt aaa ttt gat aaa cca gtc
aat gta gaa agt tat ttg ttc 1467 Ile Leu Phe Lys Phe Asp Lys Pro
Val Asn Val Glu Ser Tyr Leu Phe 430 435 440 cat agc ggc aac caa gaa
cat cct gga gat att ctg cta aac aca act 1515 His Ser Gly Asn Gln
Glu His Pro Gly Asp Ile Leu Leu Asn Thr Thr 445 450 455 460 gtg gaa
gtt ttg cct ttt aag agt gaa ggt ttg gaa ata agc aaa gaa 1563 Val
Glu Val Leu Pro Phe Lys Ser Glu Gly Leu Glu Ile Ser Lys Glu 465 470
475 acc aaa gac aaa cga tta gaa gat ggc tat ttc aga ata gga aaa ttt
1611 Thr Lys Asp Lys Arg Leu Glu Asp Gly Tyr Phe Arg Ile Gly Lys
Phe 480 485 490 gag aat ggt gtt gca gaa gga atg gtg gat cca agt ctc
aat ccc att 1659 Glu Asn Gly Val Ala Glu Gly Met Val Asp Pro Ser
Leu Asn Pro Ile 495 500 505 tca gcc ttt cga ctt tca gtt att cag aat
tct gct gtt tgg gcc att 1707 Ser Ala Phe Arg Leu Ser Val Ile Gln
Asn Ser Ala Val Trp Ala Ile 510 515 520 ctt aat gag att cat att aaa
aaa gcc acc aac tgatcatctg agaaaccaac 1760 Leu Asn Glu Ile His Ile
Lys Lys Ala Thr Asn 525 530 535 acattttttc ctgtgaattt gttaattaaa
gatagttaag catgtatctt ttttttattt 1820 ctacttgaac actacctctt
gtgaagtcta ctgtagataa gacgattgtc atttccactt 1880 ggaaagtgaa
tctcccataa taattgtatt tgtttgaaac taagctgtcc tcagatttta 1940
acttgactca aacatttttc aattatgaca gcctgttaat atgacttgta ctattttggt
2000 attatactaa tacataagag ttgtacatat tgttacattc tttaaatttg
agaaaaacta 2060 atgttacata cattttatga agggggtact tttgaggttc
acttatttta ctatt 2115 24 535 PRT Homo sapiens 24 Met Arg Leu Arg
Asn Gly Thr Val Ala Thr Ala Leu Ala Phe Ile Thr 1 5 10 15 Ser Phe
Leu Thr Leu Ser Trp Tyr Thr Thr Trp Gln Asn Gly Lys Glu 20 25 30
Lys Leu Ile Ala Tyr Gln Arg Glu Phe Leu Ala Leu Lys Glu Arg Leu 35
40 45 Arg Ile Ala Glu His Arg Ile Ser Gln Arg Ser Ser Glu Leu Asn
Thr 50 55 60 Ile Val Gln Gln Phe Lys Arg Val Gly Ala Glu Thr Asn
Gly Ser Lys 65 70 75 80 Asp Ala Leu Asn Lys Phe Ser Asp Asn Thr Leu
Lys Leu Leu Lys Glu 85 90 95 Leu Thr Ser Lys Lys Ser Leu Gln Val
Pro Ser Ile Tyr Tyr His Leu 100 105 110 Pro His Leu Leu Lys Asn Glu
Gly Ser Leu Gln Pro Ala Val Gln Ile 115 120 125 Gly Asn Gly Arg Thr
Gly Val Ser Ile Val Met Gly Ile Pro Thr Val 130 135 140 Lys Arg Glu
Val Lys Ser Tyr Leu Ile Glu Thr Leu His Ser Leu Ile 145 150 155 160
Asp Asn Leu Tyr Pro Glu Glu Lys Leu Asp Cys Val Ile Val Val Phe 165
170 175 Ile Gly Glu Thr Asp Ile Asp Tyr Val His Gly Val Val Ala Asn
Leu 180 185 190 Glu Lys Glu Phe Ser Lys Glu Ile Ser Ser Gly Leu Val
Glu Val Ile 195 200 205 Ser Pro Pro Glu Ser Tyr Tyr Pro Asp Leu Thr
Asn Leu Lys Glu Thr 210 215 220 Phe Gly Asp Ser Lys Glu Arg Val Arg
Trp Arg Thr Lys Gln Asn Leu 225 230 235 240 Asp Tyr Cys Phe Leu Met
Met Tyr Ala Gln Glu Lys Gly Ile Tyr Tyr 245 250 255 Ile Gln Leu Glu
Asp Asp Ile Ile Val Lys Gln Asn Tyr Phe Asn Thr 260 265 270 Ile Lys
Asn Phe Ala Leu Gln Leu Ser Ser Glu Glu Trp Met Ile Leu 275 280 285
Glu Phe Ser Gln Leu Gly Phe Ile Gly Lys Met Phe Gln Ala Pro Asp 290
295 300 Leu Thr Leu Ile Val Glu Phe Ile Phe Met Phe Tyr Lys Glu Lys
Pro 305 310 315 320 Ile Asp Trp Leu Leu Asp His Ile Leu Trp Val Lys
Val Cys Asn Pro 325 330 335 Glu Lys Asp Ala Lys His Cys Asp Arg Gln
Lys Ala Asn Leu Arg Ile 340 345 350 Arg Phe Arg Pro Ser Leu Phe Gln
His Val Gly Leu His Ser Ser Leu 355 360 365 Ser Gly Lys Ile Gln Lys
Leu Thr Asp Lys Asp Tyr Met Lys Pro Leu 370 375 380 Leu Leu Lys Ile
His Val Asn Pro Pro Ala Glu Val Ser Thr Ser Leu 385 390 395 400 Lys
Val Tyr Gln Gly His Thr Leu Glu Lys Thr Tyr Met Gly Glu Asp 405 410
415 Phe Phe Trp Ala Ile Thr Pro Ile Ala Gly Asp Tyr Ile Leu Phe Lys
420 425 430 Phe Asp Lys Pro Val Asn Val Glu Ser Tyr Leu Phe His Ser
Gly Asn 435 440 445 Gln Glu His Pro Gly Asp Ile Leu Leu Asn Thr Thr
Val Glu Val Leu 450 455 460 Pro Phe Lys Ser Glu Gly Leu Glu Ile Ser
Lys Glu Thr Lys Asp Lys 465 470 475 480 Arg Leu Glu Asp Gly Tyr Phe
Arg Ile Gly Lys Phe Glu Asn Gly Val 485 490 495 Ala Glu Gly Met Val
Asp Pro Ser Leu Asn Pro Ile Ser Ala Phe Arg 500 505 510 Leu Ser Val
Ile Gln Asn Ser Ala Val Trp Ala Ile Leu Asn Glu Ile 515 520 525 His
Ile Lys Lys Ala Thr Asn 530 535 25 30 DNA Artificial Sequence
Description of Artificial Sequence Primer 25 ttctcgagat gaggctccgc
aatggaactg 30 26 24 DNA Artificial Sequence Description of
Artificial Sequence Primer 26 agaaatgtgg gcttcagggc tggc 24 27 30
DNA Artificial Sequence Description of Artificial Sequence Primer
27 ttctcgagat gaggctccgc aatggaactg 30 28 24 DNA Artificial
Sequence Description of Artificial Sequence Primer 28 agaaatgtgg
gcttcagggc tggc 24 29 30 DNA Artificial Sequence Description of
Artificial Sequence Primer 29 ttctcgagat gaggctccgc aatggaactg 30
30 24 DNA Artificial Sequence Description of Artificial Sequence
Primer 30 agaaatgtgg gcttcagggc tggc 24 31 25 DNA Artificial
Sequence Description of Artificial Sequence Primer 31 ttccatcacc
tgccacacct gctgg 25 32 24 DNA Artificial Sequence Description of
Artificial Sequence Primer 32 acaaccctca gtcagacaag gagg 24 33 24
DNA Artificial Sequence Description of Artificial Sequence Primer
33 acacccccag aaatgtgggc ttca 24 34 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 34 atgaccgagt cctccttctc
ctgc 24 35 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 35 atgcccatca ccaccgacac tccg 24 36 1724 DNA Homo
sapiens CDS (43)..(1686) 36 tgcagcctcg gccccgcggg cgcccgccgc
gcacccgagg ag atg agg ctc cgc 54 Met Arg Leu Arg 1 aat ggc acc ttc
ctg acg ctg ctg ctc ttc tgc ctg tgc gcc ttc ctc 102 Asn Gly Thr Phe
Leu Thr Leu Leu Leu Phe Cys Leu Cys Ala Phe Leu 5 10 15 20 tcg ctg
tcc tgg tac gcg gca ctc agc ggc cag aaa ggc gac gtt gtg 150 Ser Leu
Ser Trp Tyr Ala Ala Leu Ser Gly Gln Lys Gly Asp Val Val 25 30 35
gac gtt tac cag cgg gag ttc ctg gcg ctg cgc gat cgg ttg cac gca 198
Asp Val Tyr Gln Arg Glu Phe Leu Ala Leu Arg Asp Arg Leu His Ala 40
45 50 gct gag cag gag agc ctc aag cgc tcc aag gag ctc aac ctg gtg
ctg 246 Ala Glu Gln Glu Ser Leu Lys Arg Ser Lys Glu Leu Asn Leu Val
Leu 55 60 65 gac gag atc aag agg gcc gtg tca gaa agg cag gcg ctg
cga gac gga 294 Asp Glu Ile Lys Arg Ala Val Ser Glu Arg Gln Ala Leu
Arg Asp Gly 70 75 80 gac ggc aat cgc acc tgg ggc cgc cta aca gag
gac ccc cga ttg aag 342 Asp Gly Asn Arg Thr Trp Gly Arg Leu Thr Glu
Asp Pro Arg Leu Lys 85 90 95 100 ccg tgg aac ggc tca cac cgg cac
gtg ctg cac ctg ccc acc gtc ttc 390 Pro Trp Asn Gly Ser His Arg His
Val Leu His Leu Pro Thr Val Phe 105 110 115 cat cac ctg cca cac ctg
ctg gcc aag gag agc agt ctg cag ccc gcg 438 His His Leu Pro His Leu
Leu Ala Lys Glu Ser Ser Leu Gln Pro Ala 120 125 130 gtg cgc gtg ggc
cag ggc cgc acc gga gtg tcg gtg gtg atg ggc atc 486 Val Arg Val Gly
Gln Gly Arg Thr Gly Val Ser Val Val Met Gly Ile 135 140 145 ccg agc
gtg cgg cgc gag gtg cac tcg tac ctg act gac act ctg cac 534 Pro Ser
Val Arg Arg Glu Val His Ser Tyr Leu Thr Asp Thr Leu His 150 155 160
tcg ctc atc tcc gag ctg agc ccg cag gag aag gag gac tcg gtc atc 582
Ser Leu Ile Ser Glu Leu Ser Pro Gln Glu Lys Glu Asp Ser Val Ile 165
170 175 180 gtg gtg ctg atc gcc gag act gac tca cag tac act tcg gca
gtg aca 630 Val Val Leu Ile Ala Glu Thr Asp Ser Gln Tyr Thr Ser Ala
Val Thr 185 190 195 gag aac atc aag gcc ttg ttc ccc acg gag atc cat
tct ggg ctc ctg 678 Glu Asn Ile Lys Ala Leu Phe Pro Thr Glu Ile His
Ser Gly Leu Leu 200 205 210 gag gtc atc tca ccc tcc ccc cac ttc tac
cct gac ttc tcc cgc ctc 726 Glu Val Ile Ser Pro Ser Pro His Phe Tyr
Pro Asp Phe Ser Arg Leu 215 220 225 cga gag tcc ttt ggg gac ccc aag
gag aga gtc agg tgg agg acc aaa 774 Arg Glu Ser Phe Gly Asp Pro Lys
Glu Arg Val Arg Trp Arg Thr Lys 230 235 240 cag aac ctc gat tac tgc
ttc ctc atg atg tac gcg cag tcc aaa ggc 822 Gln Asn Leu Asp Tyr Cys
Phe Leu Met Met Tyr Ala Gln Ser Lys Gly 245 250 255 260 atc tac tac
gtg cag ctg gag gat gac atc gtg gcc aag ccc aac tac 870 Ile Tyr Tyr
Val Gln Leu Glu Asp Asp Ile Val Ala Lys Pro Asn Tyr 265 270 275 ctg
agc acc atg aag aac ttt gca ctg cag cag cct tca gag gac tgg 918 Leu
Ser Thr Met Lys Asn Phe Ala Leu Gln Gln Pro Ser Glu Asp Trp 280 285
290 atg atc ctg gag ttc tcc cag ctg ggc ttc att ggt aag atg ttc aag
966 Met Ile Leu Glu Phe Ser Gln Leu Gly Phe Ile Gly Lys Met Phe Lys
295 300 305 tcg ctg gac ctg agc ctg att gta gag ttc att ctc atg ttc
tac cgg 1014 Ser Leu Asp Leu Ser Leu Ile Val Glu Phe Ile Leu Met
Phe Tyr Arg 310 315 320 gac aag ccc atc gac tgg ctc ctg gac cat att
ctg tgg gtg aaa gtc 1062 Asp Lys Pro Ile Asp Trp Leu Leu Asp His
Ile Leu Trp Val Lys Val 325 330 335 340 tgc aac ccc gag aag gat gcg
aag cac tgt gac cgg cag aaa gcc aac 1110 Cys Asn Pro Glu Lys Asp
Ala Lys His Cys Asp Arg Gln Lys Ala Asn 345 350 355 ctg cgg atc cgc
ttc aaa ccg tcc ctc ttc cag cac gtg ggc act cac 1158 Leu Arg Ile
Arg Phe Lys Pro Ser Leu Phe Gln His Val Gly Thr His 360 365 370 tcc
tcg ctg gct ggc aag atc cag aaa ctg aag gac aaa gac ttt gga 1206
Ser Ser Leu Ala Gly Lys Ile Gln Lys Leu Lys Asp Lys Asp Phe Gly 375
380 385 aag cag gcg ctg cgg aag gag cat gtg aac ccg cca gca gag gtg
agc 1254 Lys Gln Ala Leu Arg Lys Glu His Val Asn Pro Pro Ala Glu
Val Ser 390 395 400 acg agc ctg aag aca tac cag cac ttc acc ctg gag
aaa gcc tac ctg 1302 Thr Ser Leu Lys Thr Tyr Gln His Phe Thr Leu
Glu Lys Ala Tyr Leu 405 410 415 420 cgc gag gac ttc ttc tgg gcc ttc
acc cct gcc gcg ggg gac ttc atc 1350 Arg Glu Asp Phe Phe Trp Ala
Phe Thr Pro Ala Ala Gly Asp Phe Ile 425 430 435 cgc ttc cgc ttc ttc
caa cct cta aga ctg gag cgg ttc ttc ttc cgc 1398 Arg Phe Arg Phe
Phe Gln Pro Leu Arg Leu Glu Arg Phe Phe Phe Arg 440 445 450 agt ggg
aac atc gag cac ccg gag gac aag ctc ttc aac acg tct gtg 1446 Ser
Gly Asn Ile Glu His Pro Glu Asp Lys Leu Phe Asn Thr Ser Val 455 460
465 gag gtg ctg ccc ttc gac aac cct cag tca gac aag gag gcc ctg cag
1494 Glu Val Leu Pro Phe Asp Asn Pro Gln Ser Asp Lys Glu Ala Leu
Gln 470 475 480 gag ggc cgc acc gcc acc ctc cgg tac cct cgg agc ccc
gac ggc tac 1542 Glu Gly Arg Thr Ala Thr Leu Arg Tyr Pro Arg Ser
Pro Asp Gly Tyr 485 490 495 500 ctc cag atc ggc tcc ttc tac aag gga
gtg gca gag gga gag gtg gac 1590 Leu Gln Ile Gly Ser Phe Tyr Lys
Gly Val Ala Glu Gly Glu Val Asp 505 510 515 cca gcc ttc ggc cct ctg
gaa gca ctg cgc ctc tcg atc cag acg gac 1638 Pro Ala Phe Gly Pro
Leu Glu Ala Leu Arg Leu Ser Ile Gln Thr Asp 520 525 530 tcc cct gtg
tgg gtg att ctg agc gag atc ttc ctg aaa aag gcc gac 1686 Ser Pro
Val Trp Val Ile Leu Ser Glu Ile Phe Leu Lys Lys Ala Asp 535 540 545
taagctgcgg gcttctgagg gtaccctgtg gccagccc 1724 37 548 PRT Homo
sapiens 37 Met Arg Leu Arg Asn Gly Thr Phe Leu Thr Leu Leu Leu Phe
Cys Leu 1 5 10 15 Cys Ala Phe Leu Ser Leu Ser Trp Tyr Ala Ala Leu
Ser Gly Gln Lys 20 25 30 Gly Asp Val Val Asp Val Tyr Gln Arg Glu
Phe Leu Ala Leu Arg Asp 35 40 45 Arg Leu His Ala Ala Glu Gln Glu
Ser Leu Lys Arg Ser Lys Glu Leu 50 55 60 Asn Leu Val Leu Asp Glu
Ile Lys Arg Ala Val Ser Glu Arg Gln Ala 65 70 75 80 Leu Arg Asp Gly
Asp Gly Asn Arg Thr Trp Gly Arg Leu Thr Glu Asp 85 90 95 Pro Arg
Leu Lys Pro Trp Asn Gly Ser His Arg His Val Leu His Leu 100 105 110
Pro Thr Val Phe His His Leu Pro His Leu Leu Ala Lys Glu Ser Ser 115
120 125 Leu Gln Pro Ala Val Arg Val Gly Gln Gly Arg Thr Gly Val Ser
Val 130 135 140 Val Met Gly Ile Pro Ser Val Arg Arg Glu Val His Ser
Tyr Leu Thr 145 150 155 160 Asp Thr Leu His Ser Leu Ile Ser Glu Leu
Ser Pro Gln Glu Lys Glu 165 170 175 Asp Ser Val Ile Val Val Leu Ile
Ala Glu Thr Asp Ser Gln Tyr Thr 180 185 190 Ser Ala Val Thr Glu Asn
Ile Lys Ala Leu Phe Pro Thr Glu Ile His 195 200 205 Ser Gly Leu Leu
Glu Val Ile Ser Pro Ser Pro His Phe Tyr Pro Asp 210 215 220 Phe Ser
Arg Leu Arg Glu Ser Phe Gly Asp Pro Lys Glu Arg Val Arg 225 230 235
240 Trp Arg Thr Lys Gln Asn Leu Asp Tyr Cys Phe Leu Met Met Tyr Ala
245 250 255 Gln Ser Lys Gly Ile Tyr Tyr Val Gln Leu Glu Asp Asp Ile
Val Ala 260 265 270 Lys Pro Asn Tyr Leu Ser Thr Met Lys Asn Phe Ala
Leu Gln Gln Pro 275 280 285 Ser Glu Asp Trp Met Ile Leu Glu Phe Ser
Gln Leu Gly Phe Ile Gly 290 295 300 Lys Met Phe Lys Ser Leu Asp Leu
Ser Leu Ile Val Glu Phe Ile Leu 305 310 315 320 Met Phe Tyr Arg Asp
Lys Pro Ile Asp Trp Leu Leu Asp His Ile Leu 325 330 335 Trp Val Lys
Val Cys Asn Pro Glu Lys Asp Ala Lys His Cys Asp Arg 340 345 350 Gln
Lys Ala Asn Leu Arg Ile Arg Phe Lys Pro Ser Leu Phe Gln His 355 360
365 Val Gly Thr His Ser Ser Leu Ala Gly Lys Ile Gln Lys Leu Lys Asp
370 375 380 Lys Asp Phe Gly Lys Gln Ala Leu Arg Lys Glu His Val Asn
Pro Pro 385 390 395 400 Ala Glu Val Ser Thr Ser Leu Lys Thr Tyr Gln
His Phe Thr Leu Glu 405 410
415 Lys Ala Tyr Leu Arg Glu Asp Phe Phe Trp Ala Phe Thr Pro Ala Ala
420 425 430 Gly Asp Phe Ile Arg Phe Arg Phe Phe Gln Pro Leu Arg Leu
Glu Arg 435 440 445 Phe Phe Phe Arg Ser Gly Asn Ile Glu His Pro Glu
Asp Lys Leu Phe 450 455 460 Asn Thr Ser Val Glu Val Leu Pro Phe Asp
Asn Pro Gln Ser Asp Lys 465 470 475 480 Glu Ala Leu Gln Glu Gly Arg
Thr Ala Thr Leu Arg Tyr Pro Arg Ser 485 490 495 Pro Asp Gly Tyr Leu
Gln Ile Gly Ser Phe Tyr Lys Gly Val Ala Glu 500 505 510 Gly Glu Val
Asp Pro Ala Phe Gly Pro Leu Glu Ala Leu Arg Leu Ser 515 520 525 Ile
Gln Thr Asp Ser Pro Val Trp Val Ile Leu Ser Glu Ile Phe Leu 530 535
540 Lys Lys Ala Asp 545 38 29 DNA Artificial Sequence Description
of Artificial Sequence Primer 38 ttctcgagga gatgaggctc cgcaatggc 29
39 29 DNA Artificial Sequence Description of Artificial Sequence
Primer 39 aatctagaaa tgtgggcttc agggctggc 29 40 28 DNA Artificial
Sequence Description of Artificial Sequence Primer 40 ccctcgagat
gggggtgcac gaatgtcc 28 41 48 DNA Artificial Sequence Description of
Artificial Sequence Primer 41 ctttttgctt gttaactcct ttagtattgg
ggcgcccagg actgggag 48 42 48 DNA Artificial Sequence Description of
Artificial Sequence Primer 42 ctcccagtcc tgggcgcccc aatactaaag
gagttaacaa gcaaaaag 48 43 44 DNA Artificial Sequence Description of
Artificial Sequence Primer 43 cttccttcat tttgcaataa atgaggggcg
cccaggactg ggag 44 44 43 DNA Artificial Sequence Description of
Artificial Sequence Primer 44 atttaacttc tctcttcact gtaggggcgc
ccaggactgg gag 43 45 44 DNA Artificial Sequence Description of
Artificial Sequence Primer 45 ctcccagtcc tgggcgcccc tcatttattg
caaaatgaag gaag 44 46 43 DNA Artificial Sequence Description of
Artificial Sequence Primer 46 ctcccagtcc tgggcgcccc tacagtgaag
agagaagtta aat 43 47 26 DNA Artificial Sequence Description of
Artificial Sequence Primer 47 ttctagaatc acccttccgc aacacc 26 48 28
DNA Artificial Sequence Description of Artificial Sequence Primer
48 ttctagaatc aaggcagaac ttccaccg 28 49 38 DNA Artificial Sequence
Description of Artificial Sequence Primer 49 ttctagaatc atggttcat
gtaatcttta tccg 34 51 19 DNA Artificial Sequence Description of
Artificial Sequence Primer 51 ttcatgaggc tccgaaatg 19 52 29 DNA
Artificial Sequence Description of Artificial Sequence Primer 52
caagcttcag tttgtgactt ttttaatat 29 53 27 DNA Artificial Sequence
Description of Artificial Sequence Primer 53 tcatgatact aaaggagtta
acaagca 27 54 51 DNA Artificial Sequence Description of Artificial
Sequence Primer 54 caagcttcag tggtggtggt ggtggtggtt tgtgactttt
ttaatatgga t 51 55 18 DNA Artificial Sequence Description of
Artificial Sequence Primer 55 caagcttcag tggtggtg 18 56 32 DNA
Artificial Sequence Description of Artificial Sequence Primer 56
tcatgatatt aaaggagtta acaagcaaaa aa 32 57 51 DNA Artificial
Sequence Description of Artificial Sequence Primer 57 caagcttcag
tggtggtggt ggtggtggtt ggtggctttt ttaatatgaa t 51 58 49 DNA
Artificial Sequence Description of Artificial Sequence Primer 58
caagcttcag tggtggtggt ggtggtgtgg tttcatataa tctttatcc 49 59 19 DNA
Artificial Sequence Description of Artificial Sequence Primer 59
ttcatgaggc tccgcaatg 19 60 18 DNA Artificial Sequence Description
of Artificial Sequence Primer 60 caggttgagc tccttgga 18 61 18 DNA
Artificial Sequence Description of Artificial Sequence Primer 61
tccaaggagc tcaacctg 18 62 44 DNA Artificial Sequence Description of
Artificial Sequence Primer 62 caagcttcag tggtggtggt ggtggtggtc
ggcctttttc agga 44 63 23 DNA Artificial Sequence Description of
Artificial Sequence Primer 63 cccatgggcc gcctaacaga gga 23
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