2 International Center for Biotechnology, Osaka University, Yamada-oka 2-1, Suita-shi, Osaka 565-0871, Japan
3 Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-naka 1-1-1, Okayama 700-8530, Japan
Received on August 7, 2002; revised on October 7, 2002; accepted on October 7, 2002
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Abstract |
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Key words: extracellular / glycan synthetic pathway / N-glycan / plant suspension-cultured cell
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Introduction |
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Recently, N-glycan structures of glycoproteins from plant cells have been analyzed (Kimura et al., 1990, 1996
; Yang et al., 1996
; Shimazaki et al., 1999
). The mechanisms of N-glycan synthesis are similar between plant cells and animal cells but different in several points (Kukuruzinski and Lennon, 1998
; Lerouge et al., 1998
; Rayon et al., 1998
). In both types of cells, glycan GlcNAc2Man3- GlcNAc2 is synthesized. Then in animal cells galactose residues are added to the nonreduced end of N-glycan, with addition of sialic acids (Kukuruzinski and Lennon, 1998
; Kornfeld and Kornfeld, 1985
). In contrast, N-glycans from plant cells mostly contain ß1,2-xylosylation (Takahashi et al., 1986
) and/or
1,3-fucosylation in the core structure Man3GlcNAc2, without ß1,4-galactosylation and sialylation (Lerouge et al., 1998
; Rayon et al., 1998
). Furthermore, it was reported that these glycosylations confer immunogenicity in mammals (Wilson et al., 2001
; Ogawa et al., 1996
; Ree et al., 2000
). In the production of recombinant animal glycoproteins in plant cells, it is probable that they do not have wild-type physiological activity because of plant-type glycosylation. Thus, one of the effective techniques is to modify the plant N-glycan synthetic pathway, which allows production of glycoproteins containing glycan structures similar to those of the wild type.
Previously, we generated transgenic tobacco BY2 suspension-cultured cells with the human ß1,4- galactosyltransferase gene (GT6 cells), which showed a significant change in N-glycan structures of intracellular glycoproteins in comparison with wild-type BY2 cells (Palacpac et al., 1999a). Moreover, no
1,3-fucosylated N-glycans were detected, and only 6.6% of ß1,2-xylosylated glycans were detected. Therefore, tobacco GT6 cells are suitable for the study of production systems of recombinant animal glycoproteins.
Recently the production of effective glycoproteins has been performed using plant cells (Richter et al., 2000; Yusibov et al., 1997
; Thomas et al., 1995
; Arakawa et al., 1997
). The productions of glycoproteins, such as erythropoietin, using plant suspension-cultured cells were also reported (Matsumoto et al., 1995
; Terashima et al., 1999
; Magnuson et al., 1998
). It was shown that glycoproteins expressed in tobacco suspension-cultured cells were secreted to the spent medium (Matsumoto et al., 1995
). Therefore, secretion of glycoproteins with the expected glycan structures to the spent medium from plant suspension-cultured cells modified the glycan synthetic pathway, and in vitro modification of glycan structures from plant suspension-cultured cells may have a great advantage for the production and purification of exogenous glycoproteins with sufficient bioactivity.
We recently determined the N-glycan structures of extracellular glycoproteins from tobacco BY2 suspension-cultured cells secreted to the spent medium (Misaki et al., 2001). The results show that extracellular glycoproteins have N-glycan structures distinct from those of intracellular glycoproteins. In this report, we analyzed N-glycan structures of extracellular glycoproteins from tobacco GT6 suspension-cultured cells secreted to the spent medium and investigated the effect of the modification of the glycan synthetic pathway on the structure of N-glycans from extracellular glycoproteins. Furthermore, we performed in vitro transfer of sialic acid to extracellular glycoproteins from GT6 cells and considered the in vitro modification of glycans from plant suspension-cultured cells.
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Results |
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Structural analyses of N-glycans from tobacco GT6 spent medium
Structures of N-linked glycans are shown in Figure 2. The molecular masses of N-glycans corresponding to peak A (m/z 1354.8) and C (m/z 1355.0) agreed well with the calculated mass for GalGlcNAcMan3GlcNAc2-PA (Gal GNM3; 1354.27). ß1,4-galactosidase digestions yielded GlcNAcMan3GlcNAc2-PA (GNM3; 1192.13). Moreover, N-acetyl-ß-D-glucosaminidase digestions yielded Man3GlcNAc2-PA (M3; 988.94), and the subsequent jack bean -mannosidase digestions of glucosaminidase products yielded ManGlcNAc2-PA (M1; 664.66) as analyzed by SF-HPLC (data not shown). On a column for RP-HPLC, authentic PA sugar chain GalGN1M3 was eluted faster than GalGN1M3 (data not shown) as previously reported (Tomiya and Takahashi, 1998
). Therefore, the structure of N-glycan corresponding to peak A should be
-D-Man-(1
6)[ß-D-Gal-(1
4)-ß-D-GlcNAc-(1
2)-
-D-Man-(1
3)]ß-D-Man-(1
4)-ß-D-GlcNAc-(1
4)-GlcNAc-PA (GalGN1M3), and that corresponding to peak C should be ß-D-Gal-(1
4)-ß-D-GlcNAc-(1
2)-
-D-Man-(1
6) [
-D-Man-(1
3)] ß-D-Man-(1
4)-ß-D-GlcNAc-(1
4)-GlcNAc-PA (GalGN1 M3), as shown in Figure 2.
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The molecular mass of N-glycan corresponding to peak F (m/z 1679.8) agreed well with the calculated mass for GalGlcNAcMan5GlcNAc2-PA (GalGNM5; 1678.55). ß1, 4-galactosidase digestions yielded GlcNAcMan5GlcNAc2-PA (GNM5; 1516.41). Moreover, N-acetyl-ß-D-glucosaminidase digestions yielded Man5GlcNAc2-PA (M5; 1313.22), and the subsequent jackbean -mannosidase digestions of glucosaminidase products yielded ManGlcNAc2-PA (M1; 664.66) as analyzed by SF-HPLC (data not shown). The structure of N-glycan corresponding to peak F should be
-D-Man-(1
6)[
-D-Man-(1
3)]
-D-Man-(1
6) [ß-D-Gal-(1
4)-ß-D-GlcNAc-(1
2)-
-D-Man-(1
3)]ß-D-Man-(1
4)-ß-D-GlcNAc-(1
4)-GlcNAc-PA (GalGNM5), as shown in Figure 2.
Structures of N-linked glycans are presented in Figure 2. Other peaks were confirmed to be non-N-glycans by MS/MS analysis.
In vitro sialic acid transfer
Sialic acid was transferred to GT6 extracellular glycoproteins, BY2 extracellular glycoproteins, and asialofetuin as mediated by murine 2,6-sialyltransferase. In Sambucus nigra (SNA) lectin staining (this lectin from SNA binds sialic acid attached to terminal galactose in
2,6-linkage), glycoproteins from wild-type tobacco BY2 cells were not stained, but glycoproteins from GT6 cells, as well as asialofetuin, were detected (Figure 5). This result shows that extracellular glycoproteins from GT6 cells have sugar chains with galactose residues on the non reduced end, and that sialic acid is
2,6-sialylated to sugar chains of extracellular glycoproteins from GT6 cells.
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Discussion |
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N-glycan Gal2GN2M3 found in mammalian glycoproteins is not detectable in GT6 extracellular glycoproteins. The extension of ß1,4-linked galactose residue on the GlcNAc1,3-Man of the trimannose core structure inhibited the transfer of fucose residue by mung bean
1,3-fucosyltransferase (Leiter et al., 1999
), explaining why fewer fucosylated glycans were observed. No fucosylated N-glycans were found in GT6 extracellular glycoproteins as well as intracellular glycoproteins. However, for xylosylated N-glycans, M3X accounts for 6.6% of intracellular glycoproteins; by contrast, GalGNM3X accounts for 35.2%. For soybean ß1,2-xylosyltransferase, N-linked sugar chains with galactose residue at the reducing termini were poor substrate (Zeng et al., 1997
). Thus, in the generation of an exogenous glycoprotein production system using transgenic plant cells such as GT6 cells, xylosylation of N-glycans should be suppressed.
Sialic acid was transferred in vitro to sugar chains of extracellular glycoproteins from the GT6 spent medium. SNA lectin staining showed that sialic acid was 2,6-sialylated to sugar chains of GT6 extracellular glycoproteins, suggesting that glycans of extracellular glycoproteins from GT6 spent medium can be substrates for the synthesis of mammalian-type glycans.
In this study, we demonstrated that plant cells which synthesized the precursor of mammalian-type glycans had a possibility of in vitro modification of glycans of extracellular glycoproteins to mammalian-type glycans. Moreover, it is thought that introduction of several genes regulating the later steps of the N-glycan synthetic pathway into plant cells enables generation of useful plants with the ability for mammalian-type N-glycan synthesis. We also expect that transgenic plant cells with the mammalian-type N-glycan synthetic pathway will greatly contribute to production of biopharmaceuticals.
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Materials and methods |
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Preparation of glycoproteins from tobacco GT6 spent medium
Seven-day-old cultures of GT6 cells were centrifuged at 2000 rpm for 10 min, and the supernatant (750 ml) was collected as the GT6 spent medium. The spent medium was dialyzed against deionized water, and glycoproteins were precipitated with acetone. After centrifugation at 10,000 rpm for 20 min at 4°C, the resulting pellet (90 mg) was lyophilized and used for glycoprotein analyses.
Preparation of N-glycans and analysis by HPLC
Sugar chains were released from crude glycoproteins by hydrazinolysis (100°C for 10 h). After N-acetylation of the hydrazinolysate with saturated sodium bicarbonate and acetic anhydride, the acetylated hydrazinolysate was desalted with Dowex 50x2 (Muromachi Kagaku Kogyo Kaisha, Fukuoka, Japan), and fractionated on a TSK gel Toyopearl HW-40 (Tosoh, Tokyo, Japan) column (2.5x30 cm) in 3% acetic acid solution. The obtained oligosaccharides were pyridylaminated as previously described (Kondo et al., 1990). PA sugar chains were fractionated on Jasco 880-PU HPLC with Jasco 821-FP Intelligent Spectrofluorometer using a Cosmosil 5C18-AR column (6x250 mm, Nacalai Tesque, Kyoto, Japan) or an Asahipak NH2P-50 column (4.6x250 mm, Showa Denko, Tokyo, Japan). The concentration of PA sugar chains was monitored by measuring the fluorescence intensity at excitation and emission wavelengths of 310 nm and 380 nm, respectively.
For RP-HPLC using the Cosmosil 5C18-AR column, the PA sugar chains were eluted by increasing the acetonitrile concentration in 0.02% trifluoroacetic acid linearly from 0% to 6% for 40 min at a flow rate of 1.2 ml/min. For SF-HPLC using the Asahipak NH2P-50 column, the PA sugar chains were eluted by increasing the water content in the water-acetonitrile mixture from 26% to 50% linearly for 25 min at a flow rate of 0.7 ml/min.
Glycosidase digestions and structure analyses
All glycosidase digestions were performed using ß1,4-galactosidase (Diplococcus pneumoniae, Roche Diagnostics, (Mannheim, Germany), N-acetyl-ß-D-glucosaminidase (D. pneumoniae, Roche), or -mannosidase ( jack bean, Sigma, St. Louis, MO). For the diplococcal ß1,4-galactosidase digestions, 100 µl of a reaction mixture (0.1 M sodium acetate buffer [pH 5.5], 100 pmol PA sugar chains, and 5 mU ß1,4-galactosidase) was incubated for 2 days at 37°C. For the diplococcal N-acetyl-ß-D-glucosaminidase digestions, 100 µl of a reaction mixture (0.1 M sodium acetate buffer [pH 5.5], 100 pmol PA sugar chains, and 5 mU N-acetyl-ß-D-glucosaminidase) were incubated for 2 days at 37°C. For
-mannosidase digestions, 100 µl of a reaction mixture (50 mM sodium acetate buffer [pH 3.88], 10 mM zinc acetate, 100 pmol PA sugar chains, and 10 µU of
-mannosidase) were incubated for 2 days at 37°C. The reactions were stopped by boiling the mixtures for 3 min, and after centrifugation of the mixtures at 12,000 rpm for 10 min at room temperature, the supernatants were analyzed by SF-HPLC. In the analyses of the glycosidase digests, the PA sugar chains were eluted by increasing the water content in the water-acetonitrile mixture from 26% to 50% linearly at a flow rate of 0.7 ml/min, and their elution positions were compared with those of authentic sugar chains that were prepared previously (Palacpac et al., 1999a
) or purchased (TaKaRa Shuzo, Shiga, Japan).
Ion-spray MS
A Perkin Elmer Sciex API-III, triple-quadrupole mass spectrometer equipped with an atmospheric-pressure ionization ion source was used for MS/MS analysis. It was operated in the positive mode at an ion spray voltage of 4200 V. Samples were typically dissolved in 50% acetonitrile/water containing 0.05% formic acid at a concentration of approximately 5 pmol/µ and introduced into the electrospray needle by mechanical infusion through a microsyringe at a flow rate of 10 µl/min. The collision-activated dissociation spectrum was measured with argon as the collision gas at a collision energy of 60100 eV. Scanning was performed at a step size of 0.5 Da, and the spectrum was recorded from m/z 200.
In vitro sialic acid transfer
A reaction mixture (60 µl; 12.5 mM sodium cacodylate buffer [pH 6.0], 1 mg/ml bovine serum albumin, 0.5% Triton CF-54, 2 µM CMP-NeuAc, 6 mU 2,6-sialyltransferase [from rat liver, Wako, Osaka, Japan], and 400 µg glycoproteins from the GT6 spent medium) was incubated for 5 h at 37°C. Glycoproteins from the BY2 spent medium were used as the negative control, and asialofetuin from fetal calf serum (Sigma) was used as the positive control.
Lectin blotting
The in vitro sialyltransferase reaction mixture was separated in 12.5% sodium dodecyl sulfatepolyacrylamide gel electrophoresis under reducing conditions as previously described (Laemmli, 1970), and separated products were then transferred onto a nitrocellulose membrane. The membrane was washed with phosphate buffered saline buffer (140 mM NaCl2, 2.6 mM KCl, 20 mM Na2HPO4, and 1.5 mM KH2PO4) containing 0.05% Tween 20 and incubated with horseradish peroxidaseconjugated SNA lectin.
2,6-Sialylated glycoproteins were visualized using a POD immunostain kit (Wako).
1 To whom correspondence should be addressed; e-mail:fujiyama{at}icb.osaka-u.ac.jp
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Abbreviations |
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References |
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