U.S. patent application number 11/810567 was filed with the patent office on 2008-05-29 for method for manufacturing glycoproteins having human-type glycosylation.
This patent application is currently assigned to DFB Biotech, Inc.. Invention is credited to Kazuhito Fujiyama, Tatsuji Seki.
Application Number | 20080124798 11/810567 |
Document ID | / |
Family ID | 18411476 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080124798 |
Kind Code |
A1 |
Seki; Tatsuji ; et
al. |
May 29, 2008 |
Method for manufacturing glycoproteins having human-type
glycosylation
Abstract
The present invention provides a method for manufacturing a
glycoprotein having a human-type sugar chain comprising a step in
which transformed plant cell is obtained by introducing to a plant
cell the gene of glycosyltransferase and the gene of an exogenous
glycoprotein, and a step in which the obtained transformed plant
cell is cultivated.
Inventors: |
Seki; Tatsuji; (Osaka,
JP) ; Fujiyama; Kazuhito; (Osaka, JP) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
DFB Biotech, Inc.
Fort Worth
TX
|
Family ID: |
18411476 |
Appl. No.: |
11/810567 |
Filed: |
June 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10870635 |
Jun 17, 2004 |
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11810567 |
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09857651 |
Aug 27, 2001 |
6998267 |
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PCT/JP99/06881 |
Dec 8, 1999 |
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10870635 |
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Current U.S.
Class: |
435/419 |
Current CPC
Class: |
C12N 9/0065 20130101;
C12N 9/1051 20130101; C12N 15/8245 20130101; C12Y 204/01038
20130101; C12P 21/005 20130101; C12N 15/8257 20130101; C12N 15/8242
20130101; C12Y 111/01007 20130101 |
Class at
Publication: |
435/419 |
International
Class: |
C12N 5/10 20060101
C12N005/10 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 9, 1998 |
JP |
10-350584 |
Claims
1. A plant cell comprising a gene encoding a glycosyltransferase
and a gene encoding an exogenous glycoprotein, in which the
exogenous glycoprotein is expressed and the expressed glycoprotein
comprises a human-type sugar chain.
2. The plant cell according to claim 1, wherein the
glycosyltransferase is galactosyltransferase that transfers a
galactose residue to a non-reducing terminal acetylglucosamine
residue.
3. The plant cell according to claim 2, wherein the glycoprotein
with a human-type sugar chain comprises a core sugar chain and an
outer sugar chain, wherein the core sugar chain comprises a
plurality of mannose and acetylglucosamine, and wherein the outer
sugar chain contains a terminal sugar chain portion with a
non-reducing terminal galactose.
4. The plant cell according to claim 3, wherein the outer sugar
chain has a straight chain configuration.
5. The plant cell according to claim 3, wherein the outer sugar
chain has a branched chain configuration.
6. The plant cell according to claim 5, wherein the branched sugar
chain portion has a mono-, bi-, tri, or tetra-configuration.
7. The plant cell according to claim 3, wherein the glycoprotein
produced contains no fucose or xylose linked to one or more of the
core sugar chain, the outer sugar chain and the terminal sugar
chain.
8. The plant cell according to claim 1, wherein the gene encoding a
glycosyl transferase encodes human galactosyltransferase, human
galactosidase or human 3-galactosidase and the gene encodes an
exogenous glycoprotein that is an enzyme, a hormone, a cytokine, an
antibody, a vaccine, a receptor or a serum protein.
9. The plant cell according to claim 8, wherein the exogenous
glycoprotein encoded by the introduced gene is horseradish
peroxidase, kinase, glucocerebrosidase, .alpha.-galactosidase,
tissue-type plasminogen activator (TPA), or
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase.
10. The plant according to claim 8, wherein the exogenous
glycoprotein encoded by the introduced gene is enkephalin,
interferon-alpha, granulocyte-macrophage colony stimulating factor
(GM-CSF), granulocyte colony stimulating factor (C-CSF), chorion,
stimulating hormone, interleukin-2, interferon-beta,
interferon-gamma, erythropoietin, vascular endothelial growth
factor, human choriogonadotropin (HCG), leuteinizing hormone (LH),
thyroid stimulating hormone (TSH), prolactin, or ovary stimulating
hormone.
11. The plant cell according to claim 8, wherein the exogenous
glycoprotein encoded by the introduced gene is immunoglobulin G
(IgG) or single chain variable region antibody fragments
(scFV).
12. A plant cell comprising a glycoprotein comprising a human-type
sugar chain, comprising no fucose or xylose linked to one or more
of the core sugar chain, the outer sugar chain and the terminal
sugar chain.
13. A plant cell comprising an exogenous glycoprotein that
comprises a galactose residue linked to an N-acetylglucosamine
residue by a beta 1,4 linkage.
14. A plant cell comprising an exogenous glycoprotein that
comprises a galactose residue linked through the activity of a
galactosyltransferase.
Description
TECHNICAL FIELD
[0001] The present invention relates to expression of exogenous
glycoproteins by plants.
BACKGROUND ART
[0002] Many of the functional proteins in living organisms are
glycoproteins. It has been elucidated that the diversity of the
sugar chains in glycoproteins play several important roles
physiologically (Lain, R-A., Glycobiology, 4, 759-767, 1994).
[0003] In recent years, it has also become clear that the action of
sugar chains can be divided into two categories. In the first case,
sugar chains have a direct function as ligands for binding cells,
or as receptors for bacteria and viruses, in the clearance of
glycoproteins from the blood, lysosome targeting of lysosome
enzymes and the targeting by glycoproteins toward specific tissues
and organs. For example, the contribution of glycoprotein sugar
chains in the infection of target cells by the AIDS virus (HIV) has
been established (Rahebi, L. et al., Glycoconj. J., 12, 7-16,
1995). The surface of HIV is covered with envelope protein gp120.
The binding of gp120 sugar chains to the CD4 of target cells is the
beginning of infection by the HIV virus. In the second case, the
sugar chain itself is not the functional molecule but indirectly
contributes to the formation of the higher-order structure of
proteins, solubility of proteins, protease resistance of proteins,
inhibition of antigenicity, protein function modification, protein
regeneration rate adjustment, and adjustment of the amount of
proteins expressed in cell layers. For example, sugar chains are
instrumental in the adjustment of the adhesion of nerve cell
adhesion molecules which are distributed widely in the nervous
system (Edelman, G. M., Ann. Rev. Biochem., 54, 135-169, 1985).
[0004] In eukaryotes, glycoprotein sugar chains are synthesized on
lipids of the Endoplasmic reticulum as precursor sugar chains. The
sugar chain portion is transferred to the protein, then some of the
sugar residues on the protein are removed in the Endoplasmic
reticulum, and then the glycoprotein is transported to Golgi
bodies. In the Goldi bodies, after the excess sugar residues have
been removed, further sugar residues (e.g. mannose) are added and
the sugar chain is extended (Narimatsu, H., Microbiol Immunol., 38,
489-504, 1994).
[0005] More specifically, for example, Glc3Man9GlcNAc2 on dolichol
anchors is transferred to protein in the ER membrane (Moremen K.
W., Trimble, R. B. and Herscovics A., Glycobiology 1994 April;
4(2):113-25, Glycosidases of the asparagine-linked oligosaccharide
processing pathway: and Sturm, A. 1995 N-Glycosylation of plant
proteins. In: New Comprehensive Biochemistry. Glycoproteins, Vol.
29a., Montreuil, J., Schachter, H. and Vliegenthart, J. F. G.
(eds). Elsevier Science Publishers B. V., The Netherland, pp.
521-541). ER-glucosidase I and II removes three glucose units
(Sturm, A. 1995, supra; and Kaushal G. P. and Elbein A. D., 1989,
Glycoprotein processing enzymes in plants. In Methods Enzymology
179, Complex Carbohydrates Part F. Ginsburg V. (ed), Academic
Press, Inc. NY, pp. 452-475). The resulting high mannose structure
(Man9GlcNAc2) is trimmed by ER-mannosidase (Moremen K. W. et al,
supra,; and Kornfeld, R. and Kornfeld, S., Annu. Rev. Biochem. 54,
631-664, 1985; Assembly of asparagine-linked oligosaccharides). The
number of mannose residues removed varies according to the
differences in the accessibility to the processing enzymes. The
isomers Man8-, Man7-, Man6- and Man5GlcNAc2 are produced during
processing by ER-mannosidase and Mannosidase I (Kornfeld, R. and
Kornfeld, S., supra). When four mannose residues are completely
removed by Mannosidase I (Man I), the product is Man5GlcNAc2.
N-acetylglucosaminyl transferase I (GlcNAc I) transfers
N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Man5GlcNAc2,
resulting in GlcNAcMan5GlcNAc2 (Schachter, H., Narasimhan, S.,
Gleeson, P., and Vella, G., Glycosyltransferases involved in
elongation of N-glycosidically linked oligosaccharides of the
complex or N-acetylgalactosamine type. In: Methods Enzymol 98:
Biomembranes Part L. Fleischer, S., and Fleischer, B. (ed),
Academic Press, Inc. NY, pp. 98-134 pp. 98-134, 1983). Mannosidase
II (Man II) removes two mannose residues from GlcNAcMan5GlcNAc2,
yielding GlcNAcMan3GlcNAc2 (Kaushal, G. P. and Elbein, A. D.,
supra; and Kornfeld, R. and Kornfeld, S., supra). The
oligosaccharide GlcNAcMan4GlcNAc2 is used as a substrate of
N-acetylglucosaminyl transferase II (GlcNAc II) (Moremen K. W. et
al, supra,; Kaushal, G. P. and Elbein, A. D., supra; and Kornfeld,
R. and Kornfeld, S., supra). FIG. 19 summarizes the above described
structures of N-linked glycans and enzymes involved in sugar chain
modification pathway in the Endoplasmic reticulum and Goldi bodies.
In FIG. 19, .diamond. denotes glucose, .quadrature. denotes GlcNAc,
.largecircle. denotes mannose, denotes galactose, and .box-solid.
denotes sialic acid, respectively.
[0006] The sugar addition in the Golgi bodies is called terminal
sugar chain synthesis. The process differs widely among living
organisms. The sugar chain synthesis depends on the type of
eukaryote. The resulting sugar chain structure is species-specific,
and reflects the evolution of sugar adding transferase and the
Golgi bodies (Narimatsu, H., Cellular Biology, 15, 802-810,
1996).
[0007] Regarding aspargine-linked (N-linked) sugar chains; in
animals, there are high mannose-type sugar chains, complex-type
sugar chains and hybrid-type sugar chains. These structures are
shown in FIG. 1. The complex-type sugar chains in plants have
.alpha.1,3 fucose and .beta.1,2 xylose which are sugar residues
that are not found in animals (Johnson, K. D. and Chrispeels, M.
J., Plant Physiol., 84, 1301-1308, 1897, Kimura, Y. et al., Biosci.
Biotech. Biochem., 56, 215-222, 1992). In the case of N-linked
sugar chains, sialic acid has been found in animal sugar chains but
has not been found in plant sugar chains. Regarding galactose,
which is generally found in animal sugar chains, although the
presence thereof has been found in some plant sugar chains
(Takahashi, N. and Hotta, T., Biochemistry, 25, 388-395, 1986), the
examples thereof are few. The linkage-type thereof is a .beta.1,3
linkage (FEBS Lett 1997 Sep. 29, 415(2). 186-191, Identification of
the human Lewis (a) carbohydrate motif in a secretory peroxidase
from a plant cell suspension culture (Vaccinium mytillus L.)., Melo
N S, Nimtz M, Contradt H S, Fevereiro P S, Costa J; Plant J. 1997
Dec. 12(6), 1411-1417, N-glycans harboring the Lewis a epitope are
expressed at the surface of plant cells., Fitchette-Laine A C,
Gomord V, Cabanes M, Michalski J C, Saint Macary M, Foucher B,
Cavelier B, Hawes C, Lerouge P. Faye L). This linkage is different
from those found in animals.
[0008] Glycoproteins derived from humans include human
erythropoietin (EPO). In order to produce glycoproteins with sugar
chain structures similar to humans, these glycoproteins are
produced in animal host cells. However, EPO produced in animal
cells has a sugar chain structure that is different from the
natural human sugar chain structure. As a result, in vivo activity
of EPO is reduced (Takeuchi, M. et al., Proc. Natl. Acad. Sci. USA,
86, 7819-7822, 1989). The sugar chain structure in other proteins
derived from humans, such as hormones and interferon, have also
been analyzed and manufactured with the same glycosylation
limitations.
[0009] The methods used to introduce exogenous genes to plants
include the Agrobacterium method (Weising, K. et al., Annu. Rev.
Genet., 22, 421, 1988), the electroporation method (Toriyama, K. et
al., Bio/Technology, 6, 1072, 1988), and the gold particle method
(Gasser, C. G. and Fraley, R. T., Science, 244, 1293, 1989).
Albumin (Sijmons, P. C. et al., Bio/Technology, 8, 217, 1990),
enkephalin (Vandekerckhove, J. et al., Bio/Technology, 7, 929,
1989), and monoclonal antibodies (Benvenulo, E. et al., Plant Mol.
Biol., 17, 865, 1991 and Hiatt, A. et al., Nature, 342, 76, 1989)
have been manufactured in plants. Hepatitis B virus surface
antigens (HBsAg) (Mason, H. S. et al., Proc. Natl. Acad. Sci. USA.,
89, 11745, 1992) and secretion-type IgA (Hiatt, A. and Ma, J. S.
K., FEBS Lett., 307, 71, 1992) have also been manufactured in plant
cells. However, when human-derived glycoproteins are expressed in
plants, the sugar chains in the manufactured glycoproteins have
different structures than the sugar chains in the glycoproteins
produced in humans because the sugar adding mechanism in plants is
different from the sugar adding mechanism in animals. As a result,
glycoproteins do not have the original physiological activity and
may be immunogenic in humans (Wilson, I. B. H. et al., Glycobiol.,
Vol. 8, No. 7, pp. 651-661, 1998).
DISCLOSURE OF THE INVENTION
[0010] The purpose of the present invention is to solve the
problems associated with the prior art by providing plant-produced
recombinant glycoproteins with mammalian, e.g., human-type sugar
chains.
[0011] The present invention is a method of manufacturing a
glycoprotein having a human-type sugar chain comprising a step in
which a transformed plant cell is obtained by introducing to a
plant cell the gene of an enzyme capable of conducting a transfer
reaction of a galactose residue to a non-reducing terminal
acetylglucosamine residue and the gene of a exogenous glycoprotein,
and a step in which the obtained transformed plant cell is
cultivated.
[0012] In the present invention, the glycoprotein with a human-type
sugar chain can comprise a core sugar chain and an outer sugar
chain, the core sugar chain consists essentially of a plurality of
mannose and acetylglucosamine, and the outer sugar chain contains a
terminal sugar chain portion with a non-reducing terminal
galactose.
[0013] In the present invention, the outer sugar chain can have a
straight chain configuration or a branched configuration. In the
present invention, the branched sugar chain portion can have a
mono-, bi-, tri- or tetra configuration. In the present invention,
the glycoprotein can contain neither fucose nor xylose.
[0014] The present invention is also a plant cell having a sugar
chain adding mechanism which can conduct a transfer reaction of a
galactose residue to a non-reducing terminal acetylglucosamine
residue, wherein the sugar chain adding mechanism acts on a sugar
chain containing a core sugar chain and an outer sugar chain,
wherein the core sugar chain consists essentially of a plurality of
mannose and acetylglucosamine, and wherein the outer sugar chain
contains a terminal sugar chain portion with a non-reducing
terminal galactose.
[0015] In the present invention, a glycoprotein with a human-type
sugar chain is obtained using this method.
BRIEF DESCRIPTION OF DRAWINGS
[0016] FIG. 1. A schematic drawing of typical N-linked sugar chain
configurations.
[0017] FIG. 2. Schematic drawings of the cloning method for
hGT.
[0018] FIG. 3. Schematic drawings of the method used to construct
vector pGAhGT for hGT expression.
[0019] FIG. 4. A photograph showing a Southern blot analysis of a
genome of cultivated transformed tobacco cells. FIG. 4 (A) shows
electrophoresis after the genome DNA (40 .mu.g) has been digested
by EcoRI and HindIII. The numbers at the left indicate the position
of the DNA molecular weight marker.
[0020] FIG. 4 (B) shows a schematic drawing of a 2.2 kb fragment
containing a promoter, hGT and terminator, which is integrated into
the transformed cell.
[0021] FIG. 5. FIG. 5 is a photograph of the Western blotting of
immunoreactive protein from transformed tobacco BY2 cells (WT) and
wild type tobacco BY2 cells (WT). The protein was denatured,
electrophoresed on 10% SDS-PAGE, and then transferred electrically
to nitrocellulose film. The samples were as follows: lane 1=GT1
cell extract; lane 2=GT 6 cell extract; lane 3=GTB cell extract;
lane 4=GT9 cell extract; lane 5=wild type cell extract; lane 6=GT1
microsome fragment; lane 7=GT6 microsome fragment; lane 8=GT8
microsome fragment; lane 9=GT9 microsome fragment; lane 10=wild
type microsome fragment.
[0022] FIG. 6. An electrophoresis photograph showing the detection
of galactosylated glycoprotein using Ricinus communis (RCA.sub.120)
affinity chromatography. The electrophoresed gel was visualized by
silver staining. Lanes 1 and 2 show the protein from wild type BY2
cells, while Lanes 3 and 4 show the protein from transformed GT6
cells. The molecular weight is in KDa units.
[0023] FIG. 7. A photograph of Western blotting a showing the
detection of galactosylated glycoprotein using Ricinus communis
(RCA.sub.120) affinity chromatography. After the electrophoresed
gel had been blotted on a nitrocellulose membrane, this membrane
was visualized by lectin (RCA.sub.120) staining. Lanes 1 and 2 show
the protein from a wild type BY2 cell, while Lanes 3 and 4 show the
protein from transformed GT6 cells. The molecular weight is in
KDa.
[0024] FIG. 8. A photograph of a blotting in which the
galactosylated glycoprotein from Ricinus communis (RCA.sub.120)
affinity chromatography was probed with an antiserum specific to
xylose in complex-type plant glycans. Lanes 1 and 2 show the total
protein extracts from BY2 and GT6, respectively, and Lane 3 shows
the glycoprotein from GT6 after RCA.sub.120 affinity
chromatography. The molecular weight is in KDa units.
[0025] FIG. 9. A schematic drawing of a plasmid pBIHm-HRP which is
a binary vector with a kanamycin-resistant gene and a
hygromycin-resistant gene, and has a HRP cDNA.
[0026] FIG. 10. Photographs of isoelectric focusing and Western
blotting which show HRP production in a suspension culture of
transgenic cells. FIG. 10 (A) shows the results of isoelectric
focusing and FIG. 10 (B) shows the results of Western blotting. The
abbreviations are as follows: WT=wild-type; BY2-HRP 1, 5 and 7=the
clone numbers for BY2 cells transformed with a HRP gene; and
GT-6-HRP 4, 5 and 8=the clone numbers for GT6 cells transformed
with a HRP gene.
[0027] FIG. 11. A graph showing the reverse-phase HPLC pattern of a
PA sugar chain eluted in 0-15% acetonitrile linear gradient in
0.02% TFA over 60 minutes and at a flow rate of 1.2 ml/min. I-XI
shows the fractions eluted and purified from size-fractionation
HPLC. Excitation wavelength and emission wavelength were 310 mm and
380 mm, respectively.
[0028] FIG. 12. Graphs showing the size-fractionation HPLC pattern
of the PA sugar chain in FIG. 11. Elution was performed in a 30-50%
water gradient in the water-acetonitrile mixture over 40 minutes
and at a flow rate of 0.8 ml/min. The excitation wavelength and
emission wavelength were 310 nm and 380 nm, respectively.
[0029] FIG. 13. A graph showing the elution position of peak-K2 on
reverse phase HPLC wherein two standard sugar chain products A and
B are compared with the peak K2. The elution conditions were the
same as in FIG. 11. That is, elution was performed in 0-15%
acetonitrile linear gradient in 0.02% TFA over 60 minutes and at a
flow rate of 1.2 ml/min.
[0030] FIG. 14. Graphs showing the SF-HPLC profiles of
galactosylated PA sugar chains obtained after exoglycosidase
digestion. Elution was performed in a 30-50% water gradient in the
water-acetonitrile mixture over 25 minutes and at a flow rate of
0.8 ml/min. (A) PA-sugar chain K-2: I is the elution position of
the galactosylated PA sugar chain used; II is .beta.-galactosidase
digests of I; III is a N-acetyl-.beta.-D-glucosaminidase digests of
II; IV is jack bean .alpha.-mannosidase digests of III. (B)
PA-sugar chain L: I is the elution position of the galactosylated
PA sugar chain used; II is .beta.-galactosidase digests of I; III
is N-acetyl-.beta.-D-glucosaminidase digests of II; IV is
.alpha.1,2 mannosidase digests of III; V is jack bean
.alpha.-mannosidase digests of III.
[0031] FIG. 15. Estimated structures of the N-linked glycans
obtained from the transformed cells. The numbers in the parentheses
indicate the molar ratio.
[0032] FIG. 16. Photographs of Ricinus communes 120 agglutinin
(RCA.sub.120) affinity chromatography showing the detection of
glycosylated HRP. FIG. 16 (A) shows the results from silver
staining, and FIG. 16 (B) shows the results from lectin RCA.sub.120
staining. The lectin-stained filter was cut into strips and then
probed using lectin RCA.sub.120 pre-incubated with buffer alone (I
and II) or incubated in buffer with excess galactose (III). In
(II), HRP was treated with .beta.-galactosidase from Diplococcus
pneumoniae before SDS-PAGE. Lane 1 is a collected fraction
containing BY2-HRP and Lane 2 is a collected fraction containing
GT6-HRP. The numbers to the left refer to the location and the size
(KDa) of the standard protein.
[0033] FIG. 17. A graph showing the results of reverse-phase. HPLC
of the PA sugar chains from purified HRP after RCA.sub.120 affinity
chromatography.
[0034] FIG. 18. Photographs of Western blotting showing immune
detection of plant specific complex-type glycans. The purified HRP
is fractioned by SDS-PAGE, transferred to nitrocellulose, and
confirmed with rabbit anti-HRP (A) and an antiserum which is
specific for complex-type glycans of plants (B). Lane
1=galactosylated HRP from GT6-HRP after RCA.sub.120 affinity
chromatography; Lane 2=purified HRP from BY2-HRP. The position of
the molecule size marker is shown to the left in KDa. The
galactosylated N-glycan on HRP derived from the transformant
GT6-HRP cells did not react with an antiserum which has been shown
to specifically react with .beta.1,2 xylose residue indicative of
plant N-glycans.
[0035] FIG. 19. Structures of N-linked glycans and enzymes involved
in the sugar chain modification pathway in Endoplasmic reticulum
and Goldi bodies. .diamond. denotes glucose, .quadrature. denotes
GlcNAc, .largecircle. denotes mannose, denotes galactose, and
.box-solid. denotes sialic acid, respectively.
[0036] FIG. 20. Structures of N-linked glycans and the ratio of
each N-linked glycan in GT6 cell line along with those in wild-type
BY2 cell line determined similarly. .quadrature. denotes GlcNAc,
.largecircle. denotes mannose, denotes galactose, and .box-solid.
denotes sialic acid, respectively.
[0037] FIG. 21 illustrates one of the embodiment of the present
invention. In GT6 cell line, the isomers Man7-, Man6- and
Man5GlcNAc2 were observed. Because those high-mannose type
oligosaccharides will be converted by some glycan processing
enzymes to be substrates for .beta.1,4-galactosyltransferase (Gal
T), introduction of GlcNAc I, Man I and Man II cDNAs could more
efficiently lead the oligosaccharide Man7-5GlcNAc2 to
GlcNAcMan3GlcNAc2, which can be a substrate of GalT.
[0038] FIG. 22 also illustrates another the embodiment of the
present invention. 1,4-Galactosyltransferase (Gal T) uses UDP-2
galactose as a donor substrate and GlcNAc2Man3GlcNAc2 as an
acceptor substrate. Efficient supply of UDP-galactose will enhance
the Gal T enzyme reaction and more galactosylated oligosaccharide
will be produced.
BEST MODE FOR CARRYING OUT THE INVENTION
[0039] Hereinafter, the present invention will be described in
further detail. In performing the present invention, unless
otherwise indicated, any conventional technique can be used. These
include methods for isolating and analyzing proteins as well as
immunological methods. These methods can be conducted by using
commercial kits, antibodies and markers.
[0040] The method of the present invention relates to a method of
manufacturing glycoproteins with human-type sugar chains. In this
specification, "human-type sugar chain" refers to a sugar chain
with a galactose residue linked to a N-acetylglucosamine residue.
The galactose residue in the human-type sugar chain can be the
terminal sugar chain or a sialic acid residue can be linked to the
outside of the galactose residue. Preferably, the glycoprotein of
the present invention at least has no xylose or fucose linked to
one or more of the following portions: the core sugar chain
portion, the branched sugar chain portion, or the terminal sugar
chain portion of the human-type sugar chain. More preferably,
neither xylose or fucose should be linked to any portion of the
human-type sugar chain, and ideally there should be no xylose or
fucose contained in the human-type sugar chain at all.
[0041] The plant cells can be any plant cells desired. The plant
cells can be cultured cells, cells in cultured tissue or cultured
organs, or cells in a plant. Preferably, the plant cells should be
cultured cells, or cells in cultured tissue or cultured organs.
Most preferably, the plant cells should be cells in whole plants,
or portions thereof, that produce glycoproteins with human-type
sugar chains. The type of plant used in the manufacturing method of
the present invention can be any type of plant that is used in gene
transference. Examples of types of plants that can be used in the
manufacturing method of the present invention include plants in the
families of Solanaceae, Poaeae, Brassicaceae, Rosaceae,
Leguminosae, Curcurbitaceae, Lamlaceae, Liliaceae, Chenopodiaceae
and Umbelllferae.
[0042] Examples of plants in the Solanaceae family include plants
in the Nicotiana, Solanum, Datura, Lycopersicon and Petunia genera.
Specific examples include tobacco, eggplant, potato, tomato, chili
pepper, and petunia.
[0043] Examples of plants in the Poaeae family include plants in
the Oryza, Hordenum, Secale, Saccharum, Echinochloa and Zea genera.
Specific examples include rice, barley, rye, Echinochloa
crus-galli, sorghum, and maize.
[0044] Examples of plants in the Brassicaceae family include plants
in the Raphanus, Brassica, Arabidopsis, Wasabia, and Capsella
genera. Specific examples include Japanese white radish, rapeseed,
Arabidopsis thaliana, Japanese horseradish, and Capsella
bursa-pastoris.
[0045] Examples of plants in the Rosaceae family include plants in
the Orunus, Malus, Pynus, Fragaria, and Rosa genera. Specific
examples include plum, peach, apple, pear, Dutch strawberry, and
rose.
[0046] Examples of plants in the Leguminosae family include plants
in the Glycine, Vigna, Phaseolus, Pisum, Vicia, Arachis, Trifolium,
Alfalfa, and Medicago genera. Specific examples include soybean,
adzuki bean, kidney beans, peas, fava beans, peanuts, clover, and
alfalfa.
[0047] Examples of plants in the Curcurbitaceae family include
plants in the Luffa, Curcurbita, and Cucumis genera. Specific
examples include gourd, pumpkin, cucumber, and melon.
[0048] Examples of plants in the Lamiaceae family include plants in
the Lavandula, Mentha, and Perilla genera. Specific examples
include lavender, peppermint, and beefsteak plant.
[0049] Examples of plants in the Liliaceae family include plants in
the Allium, Lilitum, and Tulipa genera. Specific examples include
onion, garlic, lily, and tulip.
[0050] Examples of plants in the Chenopodiaceae family include
plants in the Spinacia genera. A specific example is spinach.
[0051] Examples of plants in the Umbelliferae family include plants
in the Angelica, Daucus, Cryptotaenia, and Apitum genera. Specific
examples include Japanese udo, carrot, honewort, and celery.
[0052] Preferably, the plants used in the manufacturing method of
the present invention should be tobacco, tomato, potato, rice,
maize, radish, soybean, peas, alfalfa or spinach. Ideally, the
plants used in the manufacturing method of the present invention
should be tobacco, tomato, potato, maize or soybean.
[0053] In this specification, "an enzyme capable of conducting a
transfer reaction of a galactose residue to a non-reducing terminal
acetylglucosamine residue" refers to an enzyme capable of
conducting a transfer reaction of a galactose residue to a
non-reducing terminal acetylglucosamine residue produced when a
sugar chain is added after synthesis of the protein portion of the
glycoprotein in the plant cell. Specific examples of these enzymes
include galactosyltransferase, galactosidase, and
.beta.-galactosidase. These enzymes can be derived from any animal
desired. Preferably, these enzymes should be derived from a mammal,
and ideally these enzymes should be derived from a human.
[0054] In this specification, "the gene of an enzyme capable of
conducting a transfer reaction of a galactose residue to a
non-reducing terminal acetylglucosamine residue" can be a gene
which can be isolated from an animal cell using a nucleotide
sequence of an encoded enzyme well known in the art, or
commercially available genes altered for expression in plants.
[0055] In this specification, "gene" usually refers to the
structural gene portion. A control sequence such as a promoter,
operator and terminator can be linked to the gene so as to properly
express the gene in a plant.
[0056] In this specification, "exogenous glycoproteins" refers to
glycoproteins whose expression in plants is the result of genetic
engineering methodologies. Examples of these exogenous
glycoproteins include enzymes, hormones, cytokines, antibodies,
vaccines, receptors and serum proteins. Examples of enzymes include
horseradish peroxidase, kinase, glucocerebrosidase,
.alpha.-galactosidase, tissue-type plasminogen activator (TPA), and
HMG-CoA reductase. Examples of hormones and cytokines include
enkephalin, interferon alpha, GM-CSF, G-CSF, chorion stimulating
hormone, interleukin-2, interferon-beta, interferon-gamma,
erythropoietin, vascular endothelial growth factor, human
choriogonadotropin (HCG), leuteinizing hormone (LH), thyroid
stimulating hormone (TSH), prolactin, and ovary stimulating
hormone. Examples of antibodies include IgG and scFv. Examples of
vaccines include antigens such as Hepatitis B surface antigen,
rotavirus antigen, Escherichia coli enterotoxin, malaria antigen,
rabies virus G protein, and HIV virus glycoprotein (e.g., gp120).
Examples of receptors and matrix proteins include EGF receptors,
fibronectin, a1-antitrypsin, and coagulation factor VIII. Examples
of serum proteins include albumin, complement proteins,
plasminogen, corticosteroid-binding globulin, throxine-binding
globulin, and protein C.
[0057] In this specification, "genes of exogeneous glycoproteins"
refers to a gene, which can be isolated from a cell using a
nucleotide sequence of an encoded protein well known in the art, or
commercially available genes altered for expression in plants.
[0058] The gene of the enzymes capable of conducting a transfer
reaction of a galactose residue to a non-reducing terminal
acetylglucosamine residue and the genes of exogenous glycoproteins
can be introduced to the plant cells using a method well known in
the art. These genes can be introduced separately or
simultaneously. Examples of methods for introducing genes to plant
cells include the Agrobacterium method, the electroporation method
and the particle bombardment method.
[0059] Using any method well known in the art, the plant cells with
introduced genes can be tested to make sure the introduced genes
are expressed. Examples of such methods include silver staining or
augmentation, Western blotting, Northern hybridization, and enzyme
activity detection. Cells that express the introduced genes are
referred to as transformed cells.
[0060] Transformed cells, which express enzymes capable of
conducting a transfer reaction of a galactose residue to a
non-reducing terminal acetylglucosamine residue and exogenous
glycoproteins, express exogenous glycoproteins with human-type
sugar chains. In other words, the transformed cells have human-type
sugar chain adding mechanisms. By cultivating these transformed
cells, glycoproteins with human-type sugar chains can be mass
produced. Human-type glycoproteins contain core sugar chains and
outside sugar chains. The core sugar chains consist essentially of
a plurality of mannose or acetylglucosamine. The outside sugar
chains in these glycoproteins contain non-reducing terminal sugar
chain portions. The outside sugar chains can have a straight chain
configuration or a branched chain configuration. The branched sugar
chain portion has a mono-, bi-, tri- or tetra configuration. The
glycoproteins manufactured using these transformed cells ideally do
not contain any fucose or xylose.
[0061] These transformed plant cells can remain in a cultured state
or can be differentiated into specific tissues or organs.
Alternatively, they can also be generated into plants. In this
case, the transformed plant cells can be present in the entire
plant or in specific portions of the plant, such as seed, fruit,
nut, leaf, root, stem or flower of the plant.
[0062] Glycoproteins with human-type sugar chains can be
manufactured by the transformed plant cells and then be isolated or
extracted from the plant cells. The method for isolating the
glycoproteins can be any method well known in the art. The
glycoproteins of the present invention can be used in foodstuffs
while remaining inside the transformed cells, or the glycoproteins
of the present invention can be administered to animals including
humans without antigenicity because of the added human-type sugar
chains.
[0063] Hereinafter, the present invention will be described in
detail by way of illustrative, but not restrictive, examples.
EXAMPLE 1
Cloning Human .beta.1,4 Galactose Transferase Genes
[0064] .beta.1,4 Galactosyltransferase (hGT) genes (EC2.4.1.38)
have already been cloned. A primary configuration consisting of 400
amino acids has been discovered (Masri, K. A. et al., Biochem.
Biophys. Res. Commun., 157, 657-663, 1988).
(1) Primer Preparation and Template DNA
[0065] The following primers were prepared with reference to the
report by Masri et al.
TABLE-US-00001 hGT-5Eco: (Sequence ID:1)
5'-AAAGAATTCGCGATGCCAGGCGCGCGTCCCT-3' hGT-2Sal: (Sequence I.D:2)
3'-TCGATCGCAAAACCATGTGCAGCTGATG-5' hGT-7Spe: (Sequence I.D:3)
3'-ACGGGACTCCTCAGGGGCGATGATCATAA-5' hGT6Spe: (Sequence I.D:4)
5'-AAGACTAGTGGGCCCCATGCTGATTGA-3'
[0066] Human genome DNA, human placenta cDNA, and human kidney cDNA
purchased from Clontech were used as the template DNA.
(2) Cloning the hGT Gene cDNA
[0067] (i) Human genome DNA was used as the template and hGT-5Eco
and hGT-7Spe were used as the primers; (ii) Human placenta cDNA was
used as the template and hGT-2Sal and hGT6Spe were used as the
primers. The two were combined and a PCR reaction was performed
under the following conditions. Then, 0.4 kb and 0.8 kb fragments
containing hGT encoded areas were obtained.
(PCR reaction mixture) 1 .mu.l template DNA, 5 .mu.ml 10.times.PCR
buffer solution, 4 .mu.l dNTPs (200 mM), the primers (10 pmol), and
0.5 .mu.l (Takara Shuzo Co., Ltd.) Tag polymerase (or 0.2 .mu.l Tub
polymerase), water was added to make 50 .mu.l.
[0068] (PCR Reaction Conditions) First Stage: 1 cycle, denaturation
(94.degree. C.) 5 minutes, annealing (55.degree. C.) 1 minute,
extension (72.degree. C.) 2 minutes. Second Stage: 30 cycles,
denaturation (94.degree. C.) 1 minute, annealing (55.degree. C.) 1
minute, extension (72.degree. C.) 2 minutes. Third Stage: 1 cycle,
denaturation (94.degree. C.) 1 minute, annealing (55.degree. C.) 2
minutes, extension (72.degree. C.) 5 minutes.
[0069] The two fragments were combined to form hGT gene cDNA and
cloned in pBluescript II SK+ (SK). The pBluescript II SK+ (SK) was
purchased from Stratagene Co., Ltd. FIG. 2 shows the structure of a
plasmid containing hGT gene cDNA. This shows Sequence No. 5 in the
hGT gene nucleotide sequence and Sequence No. 6 in the estimated
amino acid sequence. This nucleotide sequence differed from the hGT
sequence published by Masri et al. (see above) in the following
ways: a) The nucleotides are different in that the A in Position
No. 528 is G, the C in Position No. 562 is T, and the A in Position
No. 1047 is G, however the encoded amino acid sequence is not
changed; b) Nine nucleotides at positions from Position No. 622 to
Position No. 630 are missing; c) The G in Position No. 405 is A and
the T in Position No. 408 is A. These nucleotide changes were made
during primer preparation such that the 0.4 kb fragment and 0.8 kb
fragment are connected. There are two start codons (ATG) in hGT
gene cDNA. In this experiment, however, the gene is designed such
that translation begins from the second initial codon (Position No.
37).
EXAMPLE 2
Introduction of the hGT Gene to a Cultivated Tobacco Cell
[0070] (1) It has been reported that hGT is expressed in an active
form in Escherichia coli (Aoki, D. et al., EMBO J., 9, 3171, 1990
and Nakazawa, K. et al., J. Biochem., 113, 747, 1993). In order for
a cultivated tobacco cell to express hGT, the expression vector
pGAhGT had to be structured as shown in FIG. 3. A cauliflower
mosaic virus 35S promoter (CaMV 35S promoter), which drives gene
expression constitutively in plant cells, was used as the promoter.
A kanamycin-resistance gene was used as the selection marker. The
pGAhGT was introduced to the cultivated tobacco cell by means of
Agrobacterium method.
[0071] The Agrobacterium method was performed using the triparental
mating method of Bevan et al. (Bevan, M., Nucleic Acid Res., 12,
8711, 1984). Escherichia coli DH5.alpha. (suE44, DlacU169,
(.phi.80lacZDM15), hsdR17) (Bethesda Research Laboratories Inc.:
Focus 8 (2), 9, 1986) with pGA-type plasmids (An. G., Methods
Enzymol. 153, 292, 1987) and Escherichia coli HB101 with helper
plasmid pRK2013 (Bevan, M., Nucleic Acid Res., 12, 8711, 1984) were
left standing overnight and 37.degree. C. in a 2.times.YT medium
containing 12.5 mg/l tetracycline and 50 mg/l kanamycin, and
Agrobacterium tumefaciens EHA101 was left standing over two nights
at 28.degree. C. in a 2.times.YT medium containing 50 mg/l
kanamycin and 25 mg/l chloramphenicol. Then, 1.5 ml of each
cultured medium was removed and placed into an Eppendorf tube.
After the cells of each strain were collected, the cells were
rinsed three times in an LB medium. The cells obtained in this
manner were then suspended in 100 .mu.l of a 2.times.YT medium,
mixed with three types of bacteria, applied to a 2.times.YT agar
medium, and cultivated at 28.degree. C. whereby the pGA-type
plasmids, then underwent conjugal transfer from the E. coli to the
Agrobacterium. Two days later some of the colony appearing on the
2.times.YT agar plate was removed using a platinum loop, and
applied to an LB agar plate containing 50 mg/l kanamycin, 12.5 mg/l
tetracycline, and 25 mg/l chloramphenicol. After cultivating the
contents for two days at 28.degree. C., a single colony was
selected.
[0072] Transformation of the cultivated tobacco cells was performed
using the method described in An, G., Plant Mol. Bio. Manual, A3,
1. First, 100 .mu.l of Agrobacterium EHA101 with pGA-type plasmids
cultivated for 36 hours at 28.degree. C. in an LB medium containing
12.5 mg/l tetracycline and 4 ml of a suspension of cultivated
tobacco cells Nicotiana tabacum L. cv. bright yellow 2 (Strain No.
BY-2 obtained using Catalog No. RPC1 from the Plant Cell
Development Group of the Gene Bank at the Life Science Tsukuba
Research Center), in their fourth day of cultivation, were mixed
together thoroughly in a dish and allowed to stand in a dark place
at 25.degree. C. Two days later, some of the solution was removed
from the dish and the supernatant was separated out using a
centrifuge (1000 rpm, 5 minutes). The cell pellet was introduced to
a new medium and centrifuged again. The cells were innoculated onto
a modified LS agar plate with 150-200 mg/l kanamycin and 250 mg/l
carbenicillin. This was allowed to stand in darkness at 25.degree.
C. After two to three weeks, the cells grown to the callus stage
were transferred to a new plate and clones were selected. After two
to three weeks, the clones were transferred to a 30 ml modified LS
medium with kanamycin and carbenicillin. This selection process was
repeated over about one month. Six resistant strains were randomly
selected from the resistant strains obtained in this manner (GT 1,
4, 5, 6, 8 and 9).
(2) Verification of the Introduced hGT Genes
[0073] In the resistant strains, a 2.2 kb fragment containing a
CaMV35S promoter and an hGT gene cDNA-NOS terminator in the T-DNA
was confirmed in the genomic DNA of the cultivated tobacco cells
using a Southern blot analysis. The Southern method was performed
after the genomic DNA had been prepared from the resistant strains
mentioned above and digested by EcoRI and HindIII.
[0074] The preparation of the chromosomal DNA from the cultured
tobacco cells was performed using the Watanabe method (Watanabe,
K., Cloning and Sequence, Plant Biotechnology Experiment Manual,
Nouson Bunka Co., Ltd.). First, 10 ml of the cultivated tobacco
cells were frozen using liquid nitrogen, and then ground to powder
using a mortar and pestle. About five grams of the powder was
placed in a centrifuge tube (40 ml) rapidly such that the frozen
powder did not melt and mixed with 5 ml of a 2.times.CTAB
(cetyltrimethyl ammonium bromide) solution pre-heated to 60.degree.
C. This was well mixed, slowly for 10 minutes, and then allowed to
stand at 60.degree. C. Then, 5 ml of a chloroform:isoamylalcohol
(24:1) solution was added, and the mixture was stirred into and
emulsion. The mixture was then centrifuged (2,800 rpm, 15 minutes,
room temperature). The surface layer was then transferred to a new
40 ml centrifuge tube and the extraction process was repeated using
the chloroform:isoamylalcohol (24:1) solution. After the surface
layer had been mixed with 1/10 volume of 10% CTAB, it was
centrifuged (2,800 rpm, 15 minutes, room temperature). The surface
layer was transferred to a new centrifuge tube and then mixed with
an equal volume of cold isopropanol. The thus obtained solvent
mixture was then centrifuged (4,500 rpm, 20 minutes, room
temperature). After the supernatant had been removed using an
aspirator, it was added to 5 ml of a TE buffer solution containing
1 M sodium chloride. This was completely dissolved at 55-60.degree.
C. This was mixed thoroughly with 5 ml of frozen isopropanol and
the DNA was observed. It was placed on the tip of a chip,
transferred to an Eppendorf tube (containing 80% frozen ethanol),
and then rinsed. The DNA was then rinsed in 70% ethanol and dried._
The dried pellet was dissolved in the appropriate amount of TE
buffer solution. Then, 5 ml of RNAase A (10 mg/ml) was added, and
reacted for one hour at 37.degree. C.; Composition of the
2.times.CTAB Solution: 2% CTAB, 0.1 M Tris-HCl (pH 8.0), 1.4 M
sodium chloride, 1% polyvinylpyrrolidone (PVP); composition of the
10% CTAB solution: 10% CTAB, 0.7 M sodium chloride.
[0075] The Southern blot method was performed in the following
manner:
[0076] (i) DNA Electrophoresis and Alkali Denaturation: After 40
.mu.g of the chromosomal DNA had been completely digested by the
restriction enzyme, the standard method was used, and 1.5% agarose
gel electrophoresis was performed (50 V). It was then stained with
ethidium bromide and photographed. The gel was then shaken for 20
minutes in 400 ml of 0.25 M HCl, and the liquid removed, and the
gel permeated with 400 ml of a denaturing solution (1.5 M NaCl, 0.5
M NaOH by shaking slowly for 45 minutes. Next, the liquid was
removed, 400 ml of neutral solution (1.5 M NaCl, 0.5 M Tris-HCl pH
7.4) was added, and the solution was shaken slowly for 15 minutes.
Then, 400 ml of the neutral solution was again added, and the
solution was shaken slowly again for 15 minutes. (ii) Transfer:
After electrophoresis, the DNA was transferred to a nylon membrane
(Hybond-N Amersham) using 20.times.SSC. The transfer took more than
12 hours. After the blotted membrane was allowed to dry at room
temperature for an hour, UV fixing was performed for five minutes.
20.times.SSC Composition: 3 M NaCl, 0.3 M sodium citrate. (iii) DNA
Probe Preparation: The DNA probe preparation was performed using a
Random Prime Labeling Kit (Takara Shuzo Co., Ltd.). Next, the
reaction solution was prepared in an Eppendorf tube. After the tube
was heated for three minutes to 95.degree. C., it was rapidly
cooled in ice. Then, 25 ng of the template DNA and 2 .mu.l of the
Random Primer were added to make 5 .mu.l. Then, 2.5 .mu.l 10.times.
buffer solution, 2.5 .mu.ml dNTPs, and 5 .mu.l [.alpha.-.sup.32P]
dCTP (1.85 MBq, 50 mCi) were added, and H.sub.2O was added to bring
the volume of reaction mixture to 24 .mu.l. Then, 1 .mu.l of a
Klenow fragment was added and the solution was allowed to stand for
10 minutes at 37.degree. C. It was then passed through a NAP10
column (Pharmacia Co., Ltd.) to prepare the purified DNA. After
being heated for three minutes at 95.degree. C., it was rapidly
cooled in ice, and used as a hybridization probe. (iv)
Hybridization: 0.5% (w/v) SDS was added to the following
Pre-hybridization Solution, the membrane in (ii) was immersed in
the solution, and pre-hybridization was performed for more than two
hours at 42.degree. C. Afterwards, the DNA probe prepared in (iii)
was added, and hybridization was performed for more than 12 hours
at 42.degree. C. Composition of the Pre-hybridization Solution:
5.times.SSC, 50 mM sodium phosphate, 50% (w/v) formamide,
5.times.Denhardt's solution (prepared by diluting
10.times.Denhardt's solution), 0.1% (w/v) SDS. Composition of the
100.times.Denhardt's Solution: 2% (w/v) BSA, 2% (w/v) Ficol 400, 2%
(w/v) polyvinylpyrrolidone (PVP). (v) Autoradiography: After
rinsing in the manner described below, autoradiography was
performed using the standard method. It was performed twice for 15
minutes at 65.degree. C. in 2.times.SSC and 0.1% SDS, and once for
15 minutes at 65.degree. C. in 0.1.times.SSC and 0.1% SDS.
[0077] The results of the Southern blot analysis of the genome DNA
prepared from the resistant strains are shown in FIG. 4. As shown
in FIG. 4, the presence of the hGT gene was verified in four
strains (GT1, 6, 8 and 9).
EXAMPLE 3
Analysis of the Galactosyltransferase Transformant
[0078] The cells of the transformants (GT-1, 6, 8 and 9) and
wild-type BY-2 in the fifth through seventh day's culture both were
harvested, and then suspended in extraction buffer solution (25 mM
Tris-HCl, pH 7.4; 0.25 M sucrose, 1 mM MgCl.sub.2, 50 mM KCl). The
cells were ruptured using ultrasound processing (200 W; Kaijo Denki
Co., Ltd. Japan) or homogenized. The cell extract solution and the
microsome fractions were then prepared according to the method of
Schwientek, T. et al. (Schwientek, T. and Ernst, J. F., Gene 145,
299-303, 1994). The expression of the hGT proteins was detected
using Western blotting and anti-human galactosyltransferase (GT)
monoclonal antibodies (MAb 8628; 1:5000) (Uejima, T. et al., Cancer
Res., 52, 6158-6163, 1992; Uemura, M. et al., Cancer Res., 52,
6153-6157, 1992) (provided by Professor Narimatsu Hisashi of Soka
University). Next, the blots were incubated with horseradish
peroxidase-conjugated goat anti-mouse IgG (5% skim milk 1:1000; EY
Laboratories, Inc., CA), and a calorimetric reaction using
horseradish peroxidase was performed using the POD Immunoblotting
Kit (Wako Chemicals, Osaka).
[0079] An immunoblot analysis of the complex glycans unique to
plants was performed using polyclonal antiserum against
.beta.-fructosidase in the cell walls of carrots and horseradish
peroxidase-conjugated goat anti-rabbit IgG antibodies (5% skim milk
1:1000; Sigma) (Lauriere, M. et al., Plant Physiol. 90, 1182-1188,
1989).
[0080] The .beta.1,4-galactosyltransferase activity was assayed as
a substrate using UDP-galactose and a pyridylamino (PA-) labeled
GlcNAc.sub.2Man.sub.3GlcNAC.sub.2
(GlcNAc.sub.2Man.sub.3GlcNAc.sub.2-PA) (Morita, N. et al., J.
Biochem. 103, 332-335, 1988). The enzyme reaction solution
contained 1-120 .mu.g protein, 25 mM sodium cacodylate (pH 7.4), 10
mM MnCl.sub.2, 200 mM UDP-galactose, and 100 nM
GlcNAc.sub.2Man.sub.3GlcNAc.sub.2-PA. An HPLC analysis was
performed on the reaction product using PALPAK Type R and PALPAK
Type N columns (Takara Shuzo Co., Ltd.) and the method recommended
by the manufacturer. The GlcNAc.sub.2Man.sub.3GlcNAc.sub.2-PA used
as the standard marker was used along with
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNac.sub.2-PA and two isomers of
GalGlcNAC.sub.2Man.sub.3GlcNAc.sub.2-PA purchased from Takara Shuzo
Co. Ltd. and Honen Co., Ltd.
[0081] The immunoblottings for the proteins derived from the
transformant and the wild-type cells are shown in FIG. 5. As shown
in FIG. 5, positive signals of a molecular weight of 50 kDa were
observed. This is greater than the molecular weight estimated from
the amino acid sequence (40 kDa) and is roughly equivalent to the
bovine galactosyltransferase purified from ascites and expressed in
yeast (Uemura, M. et al., Cancer Res., 52, 6153-6157, 1992;
Schwientek, T. et al., J. Biol. Chem., 271 (7), 3398-3405, 1996).
In the microsome fraction, immunoreactive bands (FIG. 5, Lanes 1,4)
stronger than those of the cell lysate (FIG. 5, Lanes 6-8) were
observed. This means that hGT is localized preferentially in the
cell. No immunoreactive bands were detected in the wild-type
cells.
[0082] The proteins in the microsome fractions of transformant GT6
and wild-type BY-2 were bound in an RCA.sub.120 agarose column
(Wako Chemicals, Osaka), and then rinsed with 15 volumes of 10 mM
ammonium acetate pH 6.0. Next, the bound proteins were eluted using
0.2 M lactose. After separation using SDS-PAGE, the proteins were
stained using silver staining (Wako Silver Staining Kit) (FIG. 6)
or lectin (FIG. 7). In the lectin staining, the membrane blots were
rinsed in a TTBS buffer solution (10 mM Tris-HCl, pH 7.4; 0.15 M
NaCl: 0.05% Tween 20) and incubated with horseradish peroxidase
labeled RCA.sub.120 (Honen Co., Ltd.). Galactosylated glycan was
then observed using a Immunoblotting Kit (Wako Chemicals, Osaka)
(FIG. 7). As shown in FIG. 7, an RCA.sub.120 binding was not
observed in the wild-type BY2 cells, and the GT6 had a glycoprotein
with galactose on the non-reducing terminus of the glycan
portion.
[0083] The protein extract from the wild-type BY2 cells and the GT6
cells as well as the GT6 proteins eluted from the RCA.sub.120
affinity chromatography were probed using polyclonal antibodies
unique to complex glycan (FIG. 8). The antiserum binds
predominantly to the .beta.1,2-xylose residue on the plant
glycoprotein (Lauriere, M. et al., Plant Physiol. 90, 1182-1188,
1989). As shown in FIG. 8, the wild-type BY2 cells (Lane 1) contain
glycoproteins that reacted with the polyclonal antiserum. GT6
contains very few glycoproteins that reacted with the polyclonal
antiserum (Lane 2). The GT6 glycoproteins eluted from RCA.sub.120
affinity chromatography did not bind to the polyclonal antiserum,
indicating that the galactosylated glycan does not contain
.beta.1,2-xylose residue (Lane 3).
EXAMPLE 4
Introduction of the Horseradish Peroxidase (HRP) Gene to the
hGT-Introduced Cultivated Tobacco Cells
[0084] Horseradish peroxidase gene was introduced to the resultant
GT6 cell line. Among the different types of plant peroxidase
horseradish peroxidase, especially HRP isozyme C, HRP (EC1.11.1.7)
has been the subject of extensive research. HRP can be used in
various enzyme reactions because of its superior stability and a
broad spectrum of substance specificity. For example, it has been
used in enzyme immunology for binding with a secondary antibody in
Western blotting. A number of horseradish peroxidase isozyme genes
have now been cloned (Fujiyama, K. et al., Eur. J. Biochem.,
173,681-687, 1988 and Fujiyama, K. et al., Gene, 89,163-169, 1990).
ClaPeroxidase (ClaPRX) which is encoded by prxCla is first
translated as a protein consisting of 353 amino acids containing an
extra peptide consisting of 30 amino acids at the N terminus and 15
amino acids at the C terminus. Then, this is processed to form a
mature enzyme with 308 amino acids (Fujiyama, K. et al., Eur. J.
Biochem., 173, 681-687, 1988). The molecular weight of ClaPRX
ranges between 42,200 and 44,000. Of this molecular weight, sugar
chains account for 22-27%, and there are eight N-linked sugar
chains (Welinder, K. G., Eur. J. Biochem., 96,483-502, 1979). The
introduction of the ClaPRX gene was performed using the binary
vector pBIHm-HRP for HRP expression shown in FIG. 9.
[0085] The pBIHm-HRP was prepared in the following manner. First, a
1.9 kbp HindIII-SacI fragment was prepared from a vector 35S-prxCla
for plant expression, which caries an HRP cDNA (Kawaoka, A. et al.,
J. Ferment. Bioeng., 78, 49-53, 1994).
[0086] The HindIII-SacI fragment contains a full length 1.1 kbp
prxCla cDNA following a 0.8 kbp CaMV35S promoter. The 1.9 kbp
HindIII-SacI fragment was inserted in the HindIII-SacI site of the
binary vector pBI101HmB (Akama, K. et al., Plant Cell Rep., 12,
7-11, 1992). The BamHI site at 3' of the hygromycin resistant gene
(HPT gene) had been destroyed.
[0087] Because the GT6 strain is kanamycin resistant, the
hygromycin-resistant hpt gene was used as the selection marker
(Griz, L. and Davies J., Gene, 25, 179-188, 1983). The
transformation of the GT6 strain by HRP gene was performed using
the method described in Rempel, D. H. and Nelson, L. M. (Rempel, D.
H. and Nelson, L. M., Transgenic Res. 4: 199-207, 1995). In order
to obtain HRP transformant as a control, an HRP gene was introduced
to a wild-type BY2 cell to obtain a BY2-HRP strain. The
double-transformant GT6-HRP with hGT and HRP was obtained in which
an ordinary transformation process takes place.
EXAMPLE 5
Verification of the Expression of HRP in the Cultivated
Double-Transformant Tobacco Cells
[0088] Double transformant GT6-HRP, control BY2-HRP and wild-type
(WT) cell line were examined for the expression of HRP activity
using the following method. As seen in Table 1, the HRP
gene-introduced transformant had peroxidase activity about five
times higher than the wild-type cell line.
TABLE-US-00002 TABLE 1 Specific activity Clone Number [U/mg
protein] WT-HRP-1 10.3 WT-HRP-5 11.3 WT-HRP-7 12.6 GT-HRP-4 11.1
GT-HRP-5 9.35 GT-HRP-8 9.47 Wild Type 2.49
[0089] Clone BY2-HRP obtained by introducing the HRP gene to the
wild type expressed the same degree of peroxidase activity as the
GT6-HRP double transformant with hGT and HRP.
(Peroxidase Activity Measurement)
[0090] The cultivated tobacco cells were placed into an Eppendorf
tube containing Solution D and were ruptured using a homogenizer
(Homogenizer S-203, Ikeda Rika Co., Ltd.). The supernatant was
collected after centrifugation (12,000 rpm, 20 minutes, 4.degree.
C.) and then used as the crude enzyme solution. Next, 1 ml of
Solution A, 1 ml of Solution B and 2 ml of Solution C were mixed
together, and the mixture was incubated for five minutes at
25.degree. C. The crude enzyme solution appropriately diluted with
Solution D was added to the mixture, and allowed to react for three
minutes at 25.degree. C. The reaction was stopped by the addition
of 0.5 ml of 1 N HCl, and the absorbance at 480 nm was measured. As
a control, a solution with 1 N HCl added before the introduction of
the enzyme was used.
Solution A: 1 mM o-aminophenol
Solution B: 4 mM H.sub.2O.sub.2
Solution C: 200 mM sodium phosphate buffer (pH 7.0)
Solution D: 10 mM sodium phosphate buffer (pH 6.0)
[0091] Next, in order to determine whether or not the rise in
peroxidase activity was due to the expression of HRP, activity
staining was performed after separation by gel isoelectric
focusing. The isoelectric focusing was performed using a BIO-RAD
Model 111 Mini-IEF Cell. The hydrophobic surface of the PAGE gel
support film was attached to a glass plate, and then placed on a
casting tray. The prepared gel solution was poured between the
support film and the casting tray and then photopolymerized for 45
minutes under a fluorescent lamp. The sample was applied to the
gel, and the gel was positioned so as to come into contact with
both graphite electrodes wetted with distilled water in the
electrophoretic bath. Electrophoresis was then performed for 15
minutes at 100 V. 15 minutes at 200 V and 60 minutes at 450 V.
Composition of the Gel Solution (per 1 Gel Sheet): distilled water
2.75 ml, acrylamide (25% T, 3% C) 1.0 ml, 25% glycerol 1.0 ml,
Bio-lite (40%, pH 3-10) 0.25 ml, 10% ammonium persulfate 7.5 .mu.l,
0.1% sodium riboflavin5'-phosphate 25 .mu.l, TEMED 1.5 .mu.l.
[0092] The activity staining of peroxidase was performed according
to the method of Sekine et al. (Sekine et al., Plant Cell
Technology, 6, 71-75, 1994). As shown in FIG. 10, a significant
band not found in wild-type cell line was detected in the pI 7.8
position in the BY2-HRP cell line and the GT6-HRP strain. The
results of a Western analysis using anti-HRP antibodies confirmed
the detection of a signal at the position corresponding to pI 7.8
and the expression of HRP in the double transformant GT6-HRP with
hGT and HRP.
EXAMPLE 6
Structural Analysis of the N-Linked Sugar Chains in the
Transformant GT6 Cells
(Method Used to Analyze the Sugar Chain Structure)
[0093] The N-linked sugar chains in the transformant GT6 cells were
analyzed by combining reverse-phase HPLC and size-fractionation
HPLC, performing the two-dimensional PA sugar chain mapping,
performing exoglycosidase digestion, and then performing ion spray
tandem mass spectrometry (IS-MS/MS) (Perkin Elmer Co., Ltd.).
First, the cell extract solution was delipidated with acetone,
treated with hydrazine for 12 hours at 100.degree. C., and the
sugar chain portion was released. The hydrazinolysate was
N-acetylated, desalted using the Dowex 50X2 and the Dowex 1X2 (The
Dow Chemical Co., Ltd. and its representative in Japan, Muromachi
Chemical Industry Co., Ltd.), then fractionized by using 0.1 N
ammonia and the Sephadex G-25 gel filtration column (1.8.times.180
cm) (Pharmacia Co., Ltd.). Pyridylamination was then performed as
described above. The pyridylaminated sugar chains (PA sugar chains)
were then separated using a Jasco 880-PU HPLC device with a Jasco
821-FP Intelligent Spectrophotometer (Japan Spectroscopic Co.,
Ltd.) and Cosmosil 5C18-P and Asahipak NH2P-50 columns. The elution
positions were compared with a standard either produced by the
applicant or purchased (from Wako Pure Chemical Industries, Ltd.
and Takara Shuzo Co., Ltd.).
[0094] The glycosidase digestion using
N-acetyl-.beta.-D-glucosaminidase (Diplococcus pneumoniae,
Boehringer Mannheim) or mannosidases (Jack bean, Sigma) was
performed on about 1 nmol of the PA sugar chains under the same
conditions as the method described in Kimura, Y. et al., Biosci.
Biotech. Biochem. 56 (2), 215-222, 1992. Digestion using
.beta.-galactosidase (Diplococcus pneumoniae, Boehringer Mannheim)
or Aspergillus saitoi-derived .alpha.-1,2 mannosidase (provided by
Dr. Takashi Yoshida at Tohoku University) was performed by adding 1
nmol of PA sugar chains and 200 mU 1-galactosidase or 60 .mu.g of
.alpha.-1,2 mannosidase to 50 mM of sodium acetate buffer (pH 5.5)
and incubating at 37.degree. C. After the resultant reaction
solution was boiled and the enzyme reaction was stopped, a portion
of the digested product was analyzed using size-fractionation HPLC.
The molecular weight of the digested product was analyzed using ion
spray tandem mass spectrometry (IS-MS/MS) and/or compared to the
standard sugar chain as described in Palacpac, N. Q. et al.,
Biosci. Biotech. Biochem. 63(1) 35-39, 1999 and Kimura, Y. et al.,
Biosci. Biotech. Biochem. 56 (2), 215-222, 1992.
[0095] The IS-MS/MS experiment was performed using a Perkin Elmer
Sciex API-III. It was performed in positive mode with an ion spray
voltage of 4200 V. Scanning was performed every 0.5 Da, and the m/z
was recorded from 200.
(Analysis of the Sugar Chains in the GT6 Cells)
[0096] The PA sugar chains prepared from the GT6 cells were
purified and analyzed using a combination of reverse-phase HPLC and
size-fractionation HPLC. In Fraction I at the 10-20 minute
positions in the size-fractionation HPLC (FIG. 11), no N-linked
sugar chains were eluted. This suggests that the Fraction I is a
non-absorption portion containing by-products of hydrazinolysis. In
the MS/MS analysis, no fragment ion with m/z values of 300, which
corresponds to PA-GlcNAc, was detected. Similarly, Fraction XI at
the 50-60 minute positions did not have a peak indicating elution
by the size-fractionation HPLC. Therefore, it is clear that there
were no N-linked sugar chains. The 17 peaks including A-Q shown in
FIG. 12 were all collected and purified after the analysis from
Fraction II to Fraction X in the size-fractionation HPLC (FIG. 11)
was completed.
[0097] The IS-MS/MS analysis found that seven of these peaks were
N-linked sugar chains. The following is the result from the
analysis of these peaks.
[0098] The elution positions and molecular weights of
oligosaccharides-A, -E, -H, -I, -M, -O, -P and -Q (FIG. 12) did not
correspond to those of PA sugar chain standards. In the MS/MS
analysis, the m/z values of 300 and 503, which respectively
correspond to PA-GlcNAc and PA-GlcNac.sub.2, were detected.
However, the fragment ions were not detected corresponding to
ManGlcNA.sub.2 (M1) or the trimannose core sugar chain
Man.sub.3GlcNAc.sub.2 (M3) which are generally found in N-linked
sugar chain (data not shown). Even the oligosaccharides-B, -D and
-N at the other peaks did not have fragment ions detected with an
m/z value of 300. Thus, these were not N-linked sugar chains. The
seven remaining N-linked sugar chains were then examined.
[0099] The elution positions and molecular weights of peak-C (m/z
1637.5; molar ratio 9.3%), peak-F ([M+2H] 2+m/z 819.5, [M+H]+m/z
1639; molar ratio 15.9%), and peak-G (m/z 1475.5; molar ratio
19.5%) indicated high mannose-type sugar chains
Man.sub.7GlcNAC.sub.2 (Isomer M7A and M7B) and
Man.sub.6GlcNAc.sub.2 (M6B) respectively. When digested by Jack
bean .alpha.-mannosidase, it was indicated that the N-linked sugar
chains are degraded to ManGlcNAc (M1) by size-fractionation HPLC
analysis (data not shown). In an IS-MS experiment on the digestion
product, the ion with an m/z value of 665.5 corresponding to a
calculated value of 664.66 for M1 was detected, indicating that
these N-linked sugar chains have the same structure as respective
corresponding PA sugar chain standard.
[0100] Peak-J (6.6%) had a molecular weight of 1121.5, which is
almost the same as the calculated molecular weight value of m/z
1121.05 of Man.sub.3Xyl.sub.1GlcNAc.sub.2-PA (M3X). The positions
of the fragment ions were 989.5, 827.5, 665.5, 503.3 and 300. This
does not contradict the findings that Xyl, Man, Man, Man, and
GlcNAc were released in successive order from
Man.sub.3Xyl.sub.1GlcNAc2-PA. When digested using Jack bean
.alpha.-mannosidase, the mannose residues on the non-reducing
terminus can be removed, and the two-dimensional mapping revealed
the same elution positions as those of
Man.sub.1Xyl.sub.1GlcNAc.sub.2-PA (data not shown).
[0101] The results of the analysis of the IS-MS experiment on
peak-K (13.2%) fraction revealed that this fraction contains two
types of N-linked sugar chains, one has the molecular weight of
1314.0 (1.4%) and the other has the molecular weight of 1354.5
(11.8%). This fraction was subjected to reverse-phase HPLC,
purified and analyzed. The sugar chain peak K-1 with a molecular
weight of 1314.0 had the same two-dimensional mapping and m/z value
measured as that of the sugar chain standard
Man.sub.5GlcNAc.sub.2-PA (MS). When treated using jack bean
.alpha.-mannosidase, the elution positions of the degradated
product had shifted to positions similar to those of M1 in the
two-dimensional mapping. This indicates the removal of four mannose
residues.
(Galactose-Added N-Linked Type Sugar Chains in the GT6 Cells)
[0102] The determined m/z value of 1354.5 for sugar chain peak
K-2is almost the same as the molecular weight m/z value of 1354.3
predicted for Gal.sub.1GlcNAc.sub.1Man.sub.2GlcNAc.sub.2-PA
(GalGNM3). The result of the mass spectrometry indicated that
fragment ions were derived from the parent molecules. The m/z value
of 1193.5 indicated GlcNAc.sub.1Man.sub.3GlcNAc.sub.2-PA, the m/z
value of 989.5 indicated Man.sub.3GlcNAc.sub.2-PA, the m/z value of
827.5 indicated Man.sub.2GlcNAc.sub.2-PA, the m/z value of 665
indicated ManGlcNAc.sub.2-PA, the m/z value of 503 indicated
GlcNAc.sub.2-PA, the m/z value of 336 indicated ManGlcNAc, the m/z
value of 300 indicated GlcNAc-PA, and the m/z value of 204
indicated GlcNAc. From the putative N-linked sugar chain structure,
it is considered to be either of two GalGNM3 isomers (FIG. 13). It
is either Gal .beta.4GlcNAc .beta.2Man .alpha.6(Man .alpha.3)Man
.beta.4GlcNac .beta.4GlcNAc-PA or Man .alpha.6(Gal .beta.4GlcNAc
.beta.2Man .alpha.3)Man .beta.4GlcNAc .beta.4GlcNAc-PA. The
purified PA sugar chains had reverse-phase HPLC elution positions
that were the same as the sugar chain standard Man .alpha.6 (Gal
.beta.4GlcNAc .beta.2Man .alpha.3) Man .beta.4GlcNAc
.beta.4GlcNAc-PA (FIG. 13B).
[0103] The sugar chain was treated with exoglycosidase and the
structure of the sugar chain was verified. The D. pneumoniae
.beta.-galactosidase is a Gal .beta.1,4GlcNAc linkage specific
enzyme. The digested product of the sugar chain by the enzyme was
eluted at the same position as that of the
GlcNAc.sub.1Man.sub.3GlcNAc.sub.2-PA in the size-fractionation HPLC
(FIG. 14A-II). An m/z of 1192.0 was obtained from the IS-MS/MS
analysis. These results indicate a galactose residue has been
removed from the GlcNAc on the non-reducing terminus with the
.beta.1,4 binding. When the product was digested by a
N-acetyl-.beta.-D-glucosaminidase derived from Diplococcus
pneumoniae, which is .beta.1,2 GlcNAc linkage specific (Yamashita,
K. et al., J. Biochem. 93, 135-147, 1983), the digested product was
eluted at the same position as that of the standard
Man.sub.3GlcNAc.sub.2-PA in the size-fractionation HPLC (FIG.
14A-III). When the digested product was treated with jack bean
.alpha.-mannosidase, it was eluted at the same position as that of
the standard ManGlcNAc.sub.2-PA in the size-fractionation HPLC
(FIG. 14A-IV). The sugar chain structure is shown in K-2 of FIG.
15.
[0104] The mass spectroscopy analysis of Peak L (35.5%) gave
[M+2H]2+ of 840, [M+H]+ of 1680.0, which nearly matched the
molecular weight m/z value of 1678.55 expected for
Gal.sub.1GlcNAc.sub.1Man.sub.5GlcNAc.sub.2-PA (GalGNM5). The result
of the mass spectrometry indicated fragment ions derived from the
parent molecules. The m/z value of 1313.5 indicated
MansGlcNAc.sub.2-PA, the m/z value of 1152 indicated
Man.sub.4GlcNAc.sub.2-PA, the m/z value of 989.5 indicated
Man.sub.3GlcNAc.sub.2-PA, the m/z value of 827.5 indicated
Man.sub.2GlcNAc.sub.2-PA, the m/z value of 665 indicated
ManGlcNAc.sub.2-PA, the m/z value of 503 indicated GlcNAc.sub.2-PA,
the m/z value of 336 indicated ManGlcNAc, the m/z value of 300
indicated GlcNAc-PA, and the m/z value of 204 indicated GlcNAc. The
product digested with D. pneumoniae .beta.-galactosidase was eluted
at the same position as that of
GlcNAc.sub.1Man.sub.5GlcNAc.sub.2-PA in the size-fractionation HPLC
(FIG. 14B-II). The results indicate that a galactose residue is
bound to the GlcNAc on the non-reducing terminus with the .beta.1,4
linkage. The removal of the galactose was confirmed by the
molecular weights obtained from the IS-MS/MS analysis. [M+2H] 2+
was 759 and [M+H] was 1518.0. The mass spectrometry indicated
fragments ions derived from the
GlcNAc.sub.1Man.sub.5GlcNAc.sub.2-PA with a parent signal of m/z
1518.0. The m/z value of 1314 indicated Man.sub.5GlcNAc.sub.2-PA,
the m/z value of 1152 indicated Man.sub.4GlcNAc.sub.2-PA, the m/z
value of 990 indicated Man.sub.3GlcNAc.sub.2-PA, the m/z value of
827.5 indicated Man.sub.2GlcNAc.sub.2-PA, the m/z value of 665.5
indicated Man.sub.1GlcNAc.sub.2-PA, the m/z value of 503 indicated
GlcNAc.sub.2-PA, and the m/z value of 300 indicated GlcNAc-PA. When
the GlcNAc.sub.1Man.sub.5GlcNAc.sub.2-PA was digested with an
N-acetyl-1-D-glucosaminidase derived from Diplococcus pneumoniae,
the digested product was eluted at the same position as that of the
standard Man.sub.5GlcNAc.sub.2-PA in the size-fractionation HPLC
(FIG. 14B-III). Even when treated with .alpha.-1,2 mannosidase
derived from Aspergillus saitoi, the elution position did not shift
(FIG. 14B-IV). However, when treated with jack bean
.alpha.-mannosidase, it was eluted at the same position as that of
standard Man.sub.1GlcNAc.sub.2-PA in the size-fractionation HPLC
(FIG. 14B-V). This indicates the removal of four mannose residues
in the non-reducing terminus. These results indicate that in the PA
sugar chain, none of five mannose residues are .alpha.1,2 linked to
the mannose residue which are .alpha.1,3 binding. The
exoglycosidase digestion, two-dimensional sugar chain mapping, and
IS-MS/MS analysis indicate a sugar chain structure of GalGNM5 as
shown by L in FIG. 15.
[0105] FIG. 20 summarizes the above results regarding the structure
of N-linked glycans and the ratio of each N-linked glycan in GT6
cell line along with those in wild-type BY2 cell line determined
similarly. In FIG. 20, .quadrature. denotes GlcNAc, .largecircle.
denotes mannose, denotes galactose, .quadrature. with hatched lines
therein denotes xylose, and .largecircle. with dots therein denotes
fucose respectively.
[0106] In GT6 cell line, the isomers Man7-, Man6- and Man5GlcNAc2
were observed. Because those high-mannose type oligosaccharides
will be substrates for .beta.1,4-galactosyltransferase (Gal T),
introduction of GlcNAc I, Man I and Man II cDNAs can more
efficiently lead the oligosaccharide Man7-5GlcNAc2 to
GlcNAcMan3GlcNAc2, which can be a substrate of GalT (FIG. 21).
[0107] A. thaliana cglI mutant, that lacks GnT I, can not sythesize
complex type N-glycans (von Schaewen, A., Sturm, A., O'Neill, J.,
and Chrispeels, M J., Plant Physiol., 1993 August;
102(4):1109-1118, Isolation of a mutant Arabidopsis plant that
lacks N-acetyl glucosaminyl transferase I and is unable to
synthesize Golgi-modified complex N-linked glycans).
Complementation with the human GnT I in the cglI mutant indicated
that the mammalian enzyme could contribute the plant
N-glycosylation pathway (Gomez, L. and Chrispeels, M. J., Proc.
Natl. Acad. Sci. USA 1994 March 1; 91(5):1829-1833, Complementation
of an Arabidopsis thaliana mutant that lacks complex
asparagine-linked glycans with the human cDNA encoding
N-acetylglucosaminyltransferase I.) Furthermore, GnT I cDNA
isolated from A. thaliana complemented
N-acetylglucosaminyltransferase I deficiency of CHO Lec1 cells
(Bakker, H., Lommen, A., Jotdi, W., Stiekema, W., and Bosch, D.,
Biochem. Biophys. Res. Commun., 1999 Aug. 11; 261(3):829-32, An
Arabidopsis thaliana cDNA complements the
N-acetylglucosaminyltransferase I deficiency of CHO Lec1 cells).
cDNAs encoding human Man I and Man II were isolated and sequenced
(Bause, E., Bieberich, E., Rolfs, A., Volker, C. and Schmidt, B.,
Eur J Biochem 1993 Oct. 15; 217(2):535-40, Molecular cloning and
primary structure of Man9-mannosidase from human kidney; Tremblay,
L. O., Campbell, Dyke, N. and Herscovics, A., Glycobiology 1998
June:8(6):585-95, Molecular cloning, chromosomal mapping and
tissue-specific expression of a novel human alpha 1,2-mannosidase
gene involved in N-glycan maturation; and Misago. M., Liao, Y. F.,
Kudo, S., Eto, S., Mattei, M. G., Moremen, K. W., Fukuda, M. N.,
Molecular cloning and expression of cDNAs encoding human
alpha-mannosidase II and a previously unrecognized
alpha-mannosidase IIx isozyme). Human Man I has two isozymes, Man
IA and Man IB, and the nucleotide structure of isozymes' cDNA was
shown (Bause, E., et al., and Tremblay, L. O., supra).
[0108] By transforming these cDNAs into the BY cell line, an
efficient cell line producing human-type glycoprotein, can be
obtained. .beta.1,4-Galactosyltransferase (Gal T) uses
UDP-galactose as a donor substrate and GlcNAc2Man3GlcNAc2 as an
acceptor substrate. Efficient supply of UDP-galactose will enhance
the Gal T enzyme reaction, and more galactosylated oligosaccharide
will be produced (FIG. 22).
EXAMPLE 7
Structural Analysis of the Sugar Chains on the HRP in the Double
Transformant GT6-HRP Cells
[0109] A crude cell lysate was obtained from the homogenate of 50 g
of cultured GT6-HRP cells or control BY2-HRP cells grown for seven
days, respectively. This crude cell lysate solution was applied to
a CM Sepharose FF column (1.times.10 cm) (Pharmacia Co., Ltd.)
equilibrated with 10 mM of sodium phosphate buffer (pH 6.0). After
washing the column, the eluted peroxidase was measured at an
absorbance of 403 nm. The pooled fraction was concentrated using an
ultrafilter (molecular weight cut off: 10,000, Advantec Co., Ltd.),
dialyzed against 50 mM of a sodium phosphate buffer (pH 7.0), and
then applied to an equilibrated benzhydroxaminic acid-agarose
affinity column (1.times.10 cm) (KemEn Tech, Denmark). After the
column was washed in 15 volumes of 50 mM of sodium phosphate buffer
(pH 7.0), the absorbed HRP was eluted using 0.5 M boric acid
prepared in the same buffer. The peroxidase active fraction
obtained was then pooled, dialyzed, and concentrated.
[0110] The purified HRP prepared from the double transformant
GT6-HRP cells or BY2-HRP cells was applied to a 1.times.10 cm
RCA.sub.120-agarose column. The column was then washed with 15
volumes of 10 mM ammonium acetate (pH 6.0). The absorbed proteins
were then eluted and assayed using conventional methods.
[0111] Lectin staining was then performed on the purified HRP
eluted from RCA.sub.120 affinity chromatography whose specificity
is specific to .beta.1,4 linkage galactose. The lectin RCA.sub.120
was bound to only the HRP produced by the transformed cell GT6-HRP.
Because the lectin binding was dramatically reduced by
preincubation with the galactose which can compete with the lectin
(FIG. 16b-III), the binding is carbohydrate specific. Even when the
purified HRP is pre-treated with D. pneumoniae
.beta.-galactosidase, the RCA.sub.120 binding was inhibited. These
results indicate RCA bound specifically to .beta.1,4-linked
galactose at the non-reducing end of N-linked glycan on HRP. The
absence of RCA-bound glycoproteins in the BY2-HRP cells indicates
that these cells can not transfer the .beta.1,4 linked galactose
residue to the non-reducing terminus of the HRP glycan.
[0112] Reverse-phase HPLC of PA derivatives derived from HRP
purified using RCA.sub.120 indicated that the sugar chains on the
HRP proteins purified from the GT6-HRP appear as a single peak
(FIG. 17). In the reverse phase HPLC, a Cosmosil 5C18-P column or
Asahipak NH2P column was used in a Jasco 880-PU HPLC device with a
Jasco 821-FP Intelligent Spectrofluorometer. Neither bound proteins
nor detectable peaks were observed in the HRP fractions purified
from BY2-HRP. The peak obtained from the GT6-HRP in the
size-fractionation chromatography was homogenous. The
two-dimensional mapping analysis of the peak and chromatography of
the peak at the same time with standard sugar chain indicated that
the oligosaccharide contained in the peak was
Gal.sub.1GlcNAc.sub.1Man.sub.5GlcNAc.sub.2-PA. The confirmation of
this structure was provided using continuous exoglycosidase
digestion. The standard sugar chains used were a sugar chains
prepared previously (Kimura, Y. et al., Biosci. Biotech. Biochem.
56 (2), 215-222, 1992) or purchased (Wako Pure Chemical,
Industries, Ltd. Osaka and Takara Shuzo Co., Ltd.).
[0113] The PA sugar chain digested with .beta.-galactosidase (D.
pneumoniae) matched the elution position of the standard
GlcNAc.sub.1Man.sub.5GlcNAc.sub.2-PA indicating the removal of a
galactose residue .beta.1,4-linked to a non-reducing terminal
GlcNAc. Further digestion with D. pneumoniae
N-acetyl-.beta.-D-glucosaminidase of .beta.-galactosidase-digested
products produced a sugar chain equivalent which is eluted at the
same elution position of MansGlcNAc.sub.2-PA, indicating the
removal of a GlcNAc residue .beta.1,2 linked to a non-reducing
terminal mannose residue. The removed GlcNAc residue is believed to
be linked to .alpha.1,3 mannose linked to a .beta.1,4 mannose
residue in view of the N-linked type processing route of the plant.
In order to confirm the linkage position of the GlcNAc residue,
Man.sub.5GlcNAc.sub.2-PA (MS) was incubated with .alpha.1,2
mannosidase derived from Aspergillus saitoi. As expected, an
elution position shift was not detected, confirming M5 has the
structure Man .alpha.1-6(Man .alpha.1,3) Man .alpha.1-6 (Man
.alpha.1,3) Man .beta.1,4GlcNAc .beta.1,4GlcNAc as predicted. When
the sugar chain was digested using jack bean .alpha.-mannosidase,
it was eluted at the same elution positions of known
Man.sub.1GlcNAc.sub.2-PA. Therefore, the sugar chain structure
corresponded to Man .alpha.1-6(Man .alpha.1,3)Man .alpha.1-6(Gal
.beta.1,4GlcNAc .beta.1,2Man .alpha.1,3)Man .beta.1,4GlcNAc
.beta.1,4GlcNAc (Gal.sub.1GlcNAc.sub.1Man.sub.5GlcNA.sub.2). These
results indicate that the sugar chain in GT6 cell has the structure
shown in FIG. 15 and that the sugar chain structure on an HRP
protein derived from the double transformant GT6-HRP is Man
.alpha.1-6(Man .alpha.1,3)Man .alpha.1-6(Gal .beta.1,4GlcNAc
.beta.1,2Man .alpha.1,3)Man .beta.1,4GlcNAc .beta.1,4GlcNAc
(Gal.sub.1GlcNAc.sub.1Man.sub.5GlcNA.sub.2).
[0114] Similarly, the galactosylated N-glycan on HRP derived from
the transformant GT6-HRP cells did not react with an antiserum
which has been shown to specifically react with .beta.1,2 xylose
residue indicative of plant N-glycans. This indicates that one of
the sugar residues shown to be antigenic in complex plant glycan,
i.e., xylose residue, is not present (Garcia-Casado, G. et al.,
Glycobiology 6 (4): 471, 477, 1996) (FIG. 18).
INDUSTRIAL APPLICABILITY
[0115] The present invention provides a method for manufacturing a
glycoprotein with a human-type sugar chain. It also provides plant
cells that have a sugar chain adding mechanism able to perform a
reaction in which a galactose residue is transferred to a
acetylglucosamine residue on the non-reducing terminal, wherein the
sugar chain adding mechanism is capable of joining a sugar chain
which contains a core sugar chain and an outer sugar chain, wherein
the core sugar chain consists essentially of a plurality of mannose
and acetylglucosamine, and the outer sugar chain contains a
terminal sugar chain portion containing a galactose on the
non-reducing terminal. The present invention further provides a
glycoprotein with a human-type sugar chain obtained by the present
invention. A glycoprotein with a mammalian, e.g., human-type sugar
chain of the present invention is not antigenic because the
glycosylation is a human-type. Therefore, it can be useful for
administering to animals including humans.
Sequence CWU 1
1
6131DNAArtificial SequenceDescription of Artificial Sequenceprimer
hGT-5Eco 1aaagaattcg cgatgccagg cgcgcgtccc t 31228DNAArtificial
SequenceDescription of Artificial Sequenceprimer hGT-2Sal
2tcgatcgcaa aaccatgtgc agctgatg 28329DNAArtificial
SequenceDescription of Artificial Sequenceprimer hGT-7Spe
3acgggactcc tcaggggcga tgatcataa 29427DNAArtificial
SequenceDescription of Artificial Sequenceprimer hGT-6Spe
4aagactagtg ggccccatgc tgattga 2751158DNAHomo sapiensCDS(1)..(1155)
5atg cca ggc gcg tcc cta cag cgg gcc tgc cgc ctg ctc gtg gcc gtc
48Met Pro Gly Ala Ser Leu Gln Arg Ala Cys Arg Leu Leu Val Ala Val 1
5 10 15tgc gct ctg cac ctt ggc gtc acc ctc gtt tac tac ctg gct ggc
cgc 96Cys Ala Leu His Leu Gly Val Thr Leu Val Tyr Tyr Leu Ala Gly
Arg 20 25 30gac ctg agc cgc ctg ccc caa ctg gtc gga gtc tcc aca ccg
ctg cag 144Asp Leu Ser Arg Leu Pro Gln Leu Val Gly Val Ser Thr Pro
Leu Gln 35 40 45ggc ggc tcg aac agt gcc gcc gcc atc ggg cag tcc tcc
ggg gag ctc 192Gly Gly Ser Asn Ser Ala Ala Ala Ile Gly Gln Ser Ser
Gly Glu Leu 50 55 60cgg acc gga ggg gcc cgg ccg ccg cct cct cta ggc
gcc tcc tcc cag 240Arg Thr Gly Gly Ala Arg Pro Pro Pro Pro Leu Gly
Ala Ser Ser Gln 65 70 75 80ccg cgc ccg ggt ggc gac tcc agc cca gtc
gtg gat tct ggc cct ggc 288Pro Arg Pro Gly Gly Asp Ser Ser Pro Val
Val Asp Ser Gly Pro Gly 85 90 95ccc gct agc aac ttg acc tcg gtc cca
gtg ccc cac acc acc gca ctg 336Pro Ala Ser Asn Leu Thr Ser Val Pro
Val Pro His Thr Thr Ala Leu 100 105 110tcg ctg ccc gcc tgc cct gag
gag tcc ccg cta cta gtg ggc ccc atg 384Ser Leu Pro Ala Cys Pro Glu
Glu Ser Pro Leu Leu Val Gly Pro Met 115 120 125ctg att gag ttt aac
atg cct gtg gac ctg gag ctc gtg gca aag cag 432Leu Ile Glu Phe Asn
Met Pro Val Asp Leu Glu Leu Val Ala Lys Gln 130 135 140aac cca aat
gtg aag atg ggc ggc cgc tat gcc ccc agg gac tgc gtc 480Asn Pro Asn
Val Lys Met Gly Gly Arg Tyr Ala Pro Arg Asp Cys Val145 150 155
160tct cct cac aag gtg gcc atc atc att cca ttc cgc aac cgg cag gag
528Ser Pro His Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg Gln Glu
165 170 175cac ctc aag tac tgg cta tat tat ttg cac cca gtc ctg cag
cgc cag 576His Leu Lys Tyr Trp Leu Tyr Tyr Leu His Pro Val Leu Gln
Arg Gln 180 185 190cag ctg gac tat ggc atc tat gtt atc aac cag gcg
gga gac act ata 624Gln Leu Asp Tyr Gly Ile Tyr Val Ile Asn Gln Ala
Gly Asp Thr Ile 195 200 205ttc aat cgt gct aag ctc ctc aat gtt ggc
ttt caa gaa gcc ttg aag 672Phe Asn Arg Ala Lys Leu Leu Asn Val Gly
Phe Gln Glu Ala Leu Lys 210 215 220gac tat gac tac acc tgc ttt gtg
ttt agt gac gtg gac ctc att cca 720Asp Tyr Asp Tyr Thr Cys Phe Val
Phe Ser Asp Val Asp Leu Ile Pro225 230 235 240atg aat gac cat aat
gcg tac agg tgt ttt tca cag cca cgg cac att 768Met Asn Asp His Asn
Ala Tyr Arg Cys Phe Ser Gln Pro Arg His Ile 245 250 255tcc gtt gca
atg gat aag ttt gga ttc agc cta cct tat gtt cag tat 816Ser Val Ala
Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln Tyr 260 265 270ttt
gga ggt gtc tct gct cta agt aaa caa cag ttt cta acc atc aat 864Phe
Gly Gly Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Thr Ile Asn 275 280
285gga ttt cct aat aat tat tgg ggc tgg gga gga gaa gat gat gac att
912Gly Phe Pro Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile
290 295 300ttt aac aga tta gtt ttt aga ggc atg tct ata tct cgc cca
aat gct 960Phe Asn Arg Leu Val Phe Arg Gly Met Ser Ile Ser Arg Pro
Asn Ala305 310 315 320gtg gtc ggg agg tgt cgc atg atc cgc cac tca
aga gac aag aaa aat 1008Val Val Gly Arg Cys Arg Met Ile Arg His Ser
Arg Asp Lys Lys Asn 325 330 335gaa ccc aat cct cag agg ttt gac cga
att gca cac aca aag gag aca 1056Glu Pro Asn Pro Gln Arg Phe Asp Arg
Ile Ala His Thr Lys Glu Thr 340 345 350atg ctc tct gat ggt ttg aac
tca ctc acc tac cag gtg ctg gat gta 1104Met Leu Ser Asp Gly Leu Asn
Ser Leu Thr Tyr Gln Val Leu Asp Val 355 360 365cag aga tac cca ttg
tat acc caa atc aca gtg gac atc ggg aca ccg 1152Gln Arg Tyr Pro Leu
Tyr Thr Gln Ile Thr Val Asp Ile Gly Thr Pro 370 375 380agc tag
1158Ser3856385PRTHomo sapiens 6Met Pro Gly Ala Ser Leu Gln Arg Ala
Cys Arg Leu Leu Val Ala Val 1 5 10 15Cys Ala Leu His Leu Gly Val
Thr Leu Val Tyr Tyr Leu Ala Gly Arg 20 25 30Asp Leu Ser Arg Leu Pro
Gln Leu Val Gly Val Ser Thr Pro Leu Gln 35 40 45Gly Gly Ser Asn Ser
Ala Ala Ala Ile Gly Gln Ser Ser Gly Glu Leu 50 55 60Arg Thr Gly Gly
Ala Arg Pro Pro Pro Pro Leu Gly Ala Ser Ser Gln 65 70 75 80Pro Arg
Pro Gly Gly Asp Ser Ser Pro Val Val Asp Ser Gly Pro Gly 85 90 95Pro
Ala Ser Asn Leu Thr Ser Val Pro Val Pro His Thr Thr Ala Leu 100 105
110Ser Leu Pro Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met
115 120 125Leu Ile Glu Phe Asn Met Pro Val Asp Leu Glu Leu Val Ala
Lys Gln 130 135 140Asn Pro Asn Val Lys Met Gly Gly Arg Tyr Ala Pro
Arg Asp Cys Val145 150 155 160Ser Pro His Lys Val Ala Ile Ile Ile
Pro Phe Arg Asn Arg Gln Glu 165 170 175His Leu Lys Tyr Trp Leu Tyr
Tyr Leu His Pro Val Leu Gln Arg Gln 180 185 190Gln Leu Asp Tyr Gly
Ile Tyr Val Ile Asn Gln Ala Gly Asp Thr Ile 195 200 205Phe Asn Arg
Ala Lys Leu Leu Asn Val Gly Phe Gln Glu Ala Leu Lys 210 215 220Asp
Tyr Asp Tyr Thr Cys Phe Val Phe Ser Asp Val Asp Leu Ile Pro225 230
235 240Met Asn Asp His Asn Ala Tyr Arg Cys Phe Ser Gln Pro Arg His
Ile 245 250 255Ser Val Ala Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr
Val Gln Tyr 260 265 270Phe Gly Gly Val Ser Ala Leu Ser Lys Gln Gln
Phe Leu Thr Ile Asn 275 280 285Gly Phe Pro Asn Asn Tyr Trp Gly Trp
Gly Gly Glu Asp Asp Asp Ile 290 295 300Phe Asn Arg Leu Val Phe Arg
Gly Met Ser Ile Ser Arg Pro Asn Ala305 310 315 320Val Val Gly Arg
Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn 325 330 335Glu Pro
Asn Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys Glu Thr 340 345
350Met Leu Ser Asp Gly Leu Asn Ser Leu Thr Tyr Gln Val Leu Asp Val
355 360 365Gln Arg Tyr Pro Leu Tyr Thr Gln Ile Thr Val Asp Ile Gly
Thr Pro 370 375 380Ser385
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