Plant cultured cells expressing human ß1,4-galactosyltransferase secrete glycoproteins with galactose-extended N-linked glycans

Ryo Misaki2, Yoshinobu Kimura3, Nirianne Q. Palacpac2, Shohei Yoshida2, Kazuhito Fujiyama1,2 and Tatsuji Seki2

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


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Previously, we generated transgenic tobacco BY2 suspension-cultured cells (GT6 cells) that produced human ß1,4-galactosyltransferase. In this study, we analyze the N-glycan structures of glycoproteins secreted from GT6 cells to the spent medium. The N-glycans were liberated by hydrazinolysis, and the resulting oligosaccharides were labeled with 2-aminopyridine (PA). The pyridylaminated glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by the combined use of 2D PA sugar chain mapping, MS/MS analysis, and exoglycosidase digestion. The distribution of proposed N-glycan structures of GT6-secreted glycoproteins (GalGNM5 [26.8%], GalGNM4 [18.4%], GalGNM3 [19.6%], and GalGNM3X [35.2%]) is different from that found in intracellular glycoproteins (M7A [9.3%], M7B [15.9%], M6B [19.5%], M5 [1.4%], M3X [6.6%], GalGNM5 [35.5%], and GalGNM3 [11.8%]). In vitro, sialic acid was transferred to sugar chains of extracellular glycoproteins from the GT6 spent medium. The results suggest that sugar chains of extracellular glycoproteins from the GT6 spent medium are candidates for substrates of sialic acid transfer.

Key words: extracellular / glycan synthetic pathway / N-glycan / plant suspension-cultured cell


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Recent studies have shown that plants have the potential for production of biopharmaceuticals (Richter et al., 2000Go; Yusibov et al., 1997Go; Thomas et al., 1995Go; Arakawa et al., 1997Go). Plants have several advantages for use in the production of therapeutic proteins, including reduced contamination with animal pathogens, ease of genetic engineering, economical large-scale production, and eukaryotic posttranslational modification mechanisms. In eukaryotic posttranslational modification mechanisms, the synthesis of N-linked glycan is one of the important factors for maintenance of life (Ioffe and Stanley, 1994Go; Rademacher et al., 1988Go; Asano et al., 1997Go). Sugar chains play roles in the protection against protein degradation due to blood clearance, contribute to intercellular adhesion, and are associated with folding and physiological activity of proteins (Schauer, 1985Go; Varki, 1993Go).

Recently, N-glycan structures of glycoproteins from plant cells have been analyzed (Kimura et al., 1990Go, 1996Go; Yang et al., 1996Go; Shimazaki et al., 1999Go). The mechanisms of N-glycan synthesis are similar between plant cells and animal cells but different in several points (Kukuruzinski and Lennon, 1998Go; Lerouge et al., 1998Go; Rayon et al., 1998Go). 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, 1998Go; Kornfeld and Kornfeld, 1985Go). In contrast, N-glycans from plant cells mostly contain ß1,2-xylosylation (Takahashi et al., 1986Go) and/or {alpha}1,3-fucosylation in the core structure Man3GlcNAc2, without ß1,4-galactosylation and sialylation (Lerouge et al., 1998Go; Rayon et al., 1998Go). Furthermore, it was reported that these glycosylations confer immunogenicity in mammals (Wilson et al., 2001Go; Ogawa et al., 1996Go; Ree et al., 2000Go). 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., 1999aGo). Moreover, no {alpha}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., 2000Go; Yusibov et al., 1997Go; Thomas et al., 1995Go; Arakawa et al., 1997Go). The productions of glycoproteins, such as erythropoietin, using plant suspension-cultured cells were also reported (Matsumoto et al., 1995Go; Terashima et al., 1999Go; Magnuson et al., 1998Go). It was shown that glycoproteins expressed in tobacco suspension-cultured cells were secreted to the spent medium (Matsumoto et al., 1995Go). 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., 2001Go). 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Purification of PA sugar chains from tobacco GT6 spent medium
The 2-aminylpyridine (PA) sugar chains prepared from the GT6 spent medium were purified and characterized by the combined use of reversed-phase (RP) and size-fractionation (SF) high-performance liquid chromatography (HPLC) (Figure 1). Figure 1A shows several peaks of PA derivatives analyzed by RP-HPLC. Each collected fraction (1–6) was rechromatographed by SF-HPLC (Figure 1B).




View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1. PA-derivatives from glycoproteins secreted in GT6 spent medium. (A) RP-HPLC pattern of PA sugar chains 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. 1–6, individual fractions collected and purified in SF-HPLC. (B) SF-HPLC patterns of collected fractions in A. PA sugar chains were eluted by increasing the water content in the water–acetonitrile mixture from 26% to 50% for 25 min at a flow rate of 0.7 ml/min. Excitation and emission wavelengths were 310 and 380 nm, respectively.

 
Six peaks (A–F) eluted by SF-HPLC were N-linked oligosaccharides, because other peaks gave no signals at m/z 300 (GlcNAc-PA) and 500 (GlcNAc2-PA) as determined by ion-spray mass spectrometry (MS) analysis. The structural analyses of these N-glycans are described in the following section.

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 {alpha}-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, 1998Go). Therefore, the structure of N-glycan corresponding to peak A should be {alpha}-D-Man-(1->6)[ß-D-Gal-(1->4)-ß-D-GlcNAc-(1->2)-{alpha}-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)-{alpha}-D-Man-(1->6) [{alpha}-D-Man-(1->3)] ß-D-Man-(1->4)-ß-D-GlcNAc-(1->4)-GlcNAc-PA (GalGN1 M3), as shown in Figure 2.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2. Structures of N-linked glycans obtained from GT6 spent medium. Enclosed numbers in parentheses represent molar ratios.

 
The molecular masses of N-glycans corresponding to peaks B (m/z 1486.8) and D (m/z 1487.0) agreed well with the calculated mass for GalGlcNAcMan3XylGlcNAc2-PA (GalGNM3X; 1486.38) (Figure 3A). The relevant signals obtained by tandem mass spectrometry (MS/MS) analysis of peak D could be reasonably assigned as fragment ions derived from the GalGNM3X (Figure 3B), namely, m/z 1354.0 (GalGlcNAcMan3GlcNAc2-PA), m/z 1325.0 (GlcNAcMan3XylGlcNAc2-PA), m/z 1191.0 (GlcNAc-Man3GlcNAc2-PA), m/z 1121.5 (Man3XylGlcNAc2-PA), m/z 989.5 (Man3GlcNAc2-PA), m/z 828.5 (Man2GlcNAc2-PA), m/z 503.0 (GlcNAc2-PA), and m/z 300.5 (GlcNAc-PA).




View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3. ESI-MS (A) and ESI-MS/MS (B) spectra of PA sugar chains from peak D in Figure 1B. Samples were typically dissolved in 50% acetonitrile/water containing 0.05% formic acid at a concentration of 5 pmol/µl and introduced into the electrospray needle through a microsyringe at a flow rate of 10 µl/min.

 
ß1,4-galactosidase digestions yielded GlcNAcMan3- XylGlcNAc2-PA (GNM3X; 1324.24) (Figure 4A-II). Moreover, N-acetyl-ß-D-glucosaminidase digestions yielded Man3XylGlcNAc2-PA (M3X; 1121.05) (Figure 4A-III), and the subsequent jack bean {alpha}-mannosidase digestions of glucosaminidase products yielded ManXylGlcNAc2-PA (MX; 796.77) as analyzed by SF-HPLC (data not shown). Previously, we confirmed that GalGN1M3X is eluted faster than GalGN1M3X on a column for RP-HPLC (unpublished data). Therefore, the structure of N-glycan corresponding to peak B should be {alpha}-D-Man-(1->6)[ß-D-Gal-(1->4)-ß-D-GlcNAc-(1->2)-{alpha}-D-Man-(1->3)][ß-D-Xyl-(1->2)]ß-D-Man-(1->4)-ß-D-GlcNAc-(1->4)-GlcNAc-PA (GalGN1M3X), and that corresponding to peak D should be ß-D-Gal-(1->4)-ß-D-GlcNAc-(1->2)-{alpha}-D-Man-(1->6)[{alpha}-D-Man-(1->3)] [ß-D-Xyl-(1->2)]ß-D-Man-(1-> 4)-ß-D-GlcNAc-(1->4)-GlcNAc-PA (GalGN1M3X), as shown in Figure 2.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4. SF-HPLC profiles of exoglycosidase digestion products of PA sugar chains prepared from GT6 spent medium. PA sugar chains were eluted by increasing the water content in the water–acetonitrile mixture from 26% to 50% for 25 min at a flow rate of 0.7 ml/min. Excitationand emission wavelengths were 310 and 380 nm, respectively. (A) PA-derivative from Peak B. I, native PA sugar chain; II, ß-galactosidase digest of I; III, N-acetyl-ß-D-glucosaminidase digest of II. (B) PA-derivative from Peak E. I, native PA sugar chain; II, ß-galactosidase digest of I; III, N-acetyl-ß-D-glucosaminidase digest of II.

 
The molecular mass of N-glycan corresponding to peak E (m/z 1516.6) agreed well with the calculated mass for GalGlcNAcMan4GlcNAc2-PA (GalGNM4; 1516.41). ß1,4galactosidase digestions yielded GlcNAcMan4-GlcNAc2-PA (GNM4; 1354.27) (Figure 4B-II). Moreover, N-acetyl-ß-D-glucosaminidase digestions yielded Man4-GlcNAc2-PA (M4; 1151.08) (Figure 4B-III), which can be further digested by jackbean {alpha}-mannosidase yielding ManGlcNAc2-PA (M1; 664.66) as analyzed by SF-HPLC (data not shown). The structure of N-glycan corresponding to peak E should be {alpha}-D-Man-(1->6)-{alpha}-D-Man-(1->6) [ß-D-Gal-(1->4)-ß-D-GlcNAc-(1->2)-{alpha}-D-Man-(1->3)]ß-D-Man-(1->4)-ß-D-GlcNAc-(1->4)-GlcNAc-PA (GalGNM4) or {alpha}-D-Man-(1->3)-{alpha}-D-Man-(1->6)[ß-D-Gal-(1->4)-ß-D-GlcNAc-(1->2)-{alpha}-D-Man-(1->3)]ß-D-Man-(1->4)-ß-D-GlcNAc-(1->4)-GlcNAc-PA (GalGN M4), as shown in Figure 2.

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 {alpha}-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 {alpha}-D-Man-(1->6)[{alpha}-D-Man-(1->3)]{alpha}-D-Man-(1->6) [ß-D-Gal-(1->4)-ß-D-GlcNAc-(1->2)-{alpha}-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 {alpha}2,6-sialyltransferase. In Sambucus nigra (SNA) lectin staining (this lectin from SNA binds sialic acid attached to terminal galactose in {alpha}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 {alpha}2,6-sialylated to sugar chains of extracellular glycoproteins from GT6 cells.



View larger version (102K):
[in this window]
[in a new window]
 
Fig. 5. Lectin blotting analysis of glycoproteins by using SNA lectinafter in vitro {alpha}2,6-sialic acid transfer reation. BY2, glycoproteins from wild-type BY2 spent medium after reaction; GT6, glycoproteins from GT6 spent medium after reaction; Asialofetuin, asialofetuin from fetalcalf serum after reaction. Molecular markers are in kDa.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
It was shown that transgenic tobacco GT6 suspension-cultured cells with the hGT gene had N-linked glycan structures containing galactose residues of extracellular glycoproteins as well as intracellular glycoproteins. There are seven N-glycan structures of GT6 intracellular glycoproteins; namely, M7A (9.3%), M7B (15.9%), M6B (19.5%), M5 (1.4%), M3X (6.6%), GalGNM5 (35.5%), and GalGNM3 (11.8%). On the other hand, the proposed N-glycan structures of extracellular glycoproteins are GalGNM5 (26.8%), GalGNM4 (18.4%), GalGNM3 (19.6%), and GalGNM3X (35.2%) (Table I), all of which have galactose residues that are ß1,4-linked to the nonreduced end of sugar chains (intracellular; 47.3% of total N-glycans). This result suggests that galactosylated N-glycans are secreted out of a cell through the intracellular secretory pathway, even though secretory-type extracellular glycoproteins are galactosylated. N-glycans of GT6 extracellular glycoproteins have not only GalGNM5 and GalGNM3 found on intracellular N-glycans but also GalGNM4 and GalGNM3X containing the xylose residue. Moreover, we found no extracellular or intracellular N-glycans with the fucose residue. Moreover, it was thought that the molar ratios of nongalactosylated extracellular N-glycans, if they exist, were less than the detectable level.


View this table:
[in this window]
[in a new window]
 
Table I. Comparison of sugar chain structures and molar ratios (%) of extracellular and intracellular glycoproteins

 
In previous studies, we analyzed the N-glycan structures of intracellular (Palacpac et al., 1999bGo) and extracellular (Misaki et al., 2001Go) glycoproteins produced in tobacco BY2 suspension-cultured cells. The N-glycan structures of intracellular glycoproteins from BY2 cells consist of M3FX (41.0%), GN1M3FX (21.7%), GN2M3FX (26.5%), M3X (3.3%), and M5A (7.5%). Both {alpha}1,3-fucosylated and ß1,2-xylosylated N-glycans account for 89.2%. However, there are 10 N-glycan structures of BY2 extracellular glycoproteins, namely, M3FX (8.3%), GN1M3FX (5.8%), GN1M3X (5.3%), GN1M3X (0.9%), GN2M3X (32.1%), GN2M3 (4.9%), M3X (3.7%), M7A (3.8%), M6B (6.4%), and M5A (28.8%). Both {alpha}1,3-fucosylated and ß1,2-xylosylated N-glycans account for only 12.1%. In contrast, the molar ratios of xylosylated but not fucosylated N-glycans (GNM3X and GN2M3X) are larger than those of intracellular glycoproteins. This tendency was observed in N-glycans of GT6 extracellular glycoproteins. The structure of GalGNM3X that is absent from intracellular glycoproteins accounts for 35.2%. It has been shown that {alpha}-mannosidase is found in the culture medium of suspension-cultured sycamore cells (Driouich et al., 1989Go). The activity of glycosidases released from tobacco suspension-cultured cells may also alter the N-glycan structures of extracellular glycoproteins. Thus, extracellular glycoproteins may be modified by {alpha}-mannosidase secreted from tobacco cells; as a result, GalGNM4 and GalGNM3 may be synthesized from GalGNM5.

N-glycan Gal2GN2M3 found in mammalian glycoproteins is not detectable in GT6 extracellular glycoproteins. The extension of ß1,4-linked galactose residue on the GlcNAc{alpha}1,3-Man of the trimannose core structure inhibited the transfer of fucose residue by mung bean {alpha}1,3-fucosyltransferase (Leiter et al., 1999Go), 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., 1997Go). 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 {alpha}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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Cultivation of tobacco GT6 suspension-cultured cells
Cells of the tobacco GT6 cell line were subcultured weekly in the modified Linsmaier and Skoog medium (Nagata et al., 1981Go) containing vitamins (10 mg thiamine-HCl, 1 mg pyridoxin-HCl, and 100 mg myoinositol per L) and antibiotics (250 mg carbenicillin sodium salt per L and 150 mg kanamycin sulfate per liter). The cell line was maintained by regularly transferring 4 ml of culture into 95 ml fresh medium in a 300-ml Erlenmeyer flask. Cultures were incubated in the dark at 26°C on a gyrating shaker at 120 rpm.

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., 1990Go). 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 {alpha}-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 {alpha}-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 {alpha}-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., 1999aGo) 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 60–100 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 {alpha}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 sulfate–polyacrylamide gel electrophoresis under reducing conditions as previously described (Laemmli, 1970Go), 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 peroxidase–conjugated SNA lectin. {alpha}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 Back


    Abbreviations
 
HPLC, high-performance liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PA, 2-aminopyridine; RP, reversed-phase; SF, size fractionation; SNA, Sambucus nigra.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Arakawa, T., Chong, D.K.X., Merritt, J.L., and Langridge, W.H.R. (1997) Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res., 6, 403–413.[CrossRef][ISI][Medline]

Asano, M., Furukawa, K., Kido, M., Matsumoto, S., Umesaki, Y., Kochibe, N., and Iwakura, Y. (1997) Growth retardation and early death of ß-1,4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells. EMBO J., 16, 1850–1857.[Abstract/Free Full Text]

Driouich, A., Gonnet, P., Makkie, M., Laine, A.-C., and Faye, L. (1989) The role of high-mannose and complex asparagines-linked glycans in secretion and stability of glycoproteins. Planta, 180, 96–104.[ISI]

Ioffe, E. and Stanley, P. (1994) Mice lacking N-acetyl glucosaminyltransferse I activity die at mid-gestation, revealing an essential role for complex or hybrid N-linked carbohydrates. Proc. Natl Acad. Sci. USA, 91, 728–732.[Abstract/Free Full Text]

Kimura, Y., Suehisa, H., Yamaguchi, O., Nakajima, S., and Takagi, S. (1990) Structures of sugar chains of water-soluble glycoproteins in developing caster bean cotyledons. Agric. Biol. Chem., 54, 3259–3267.[ISI][Medline]

Kimura, Y., Ohno, A., and Takagi, S. (1996) Structural elucidation of N-linked sugar chains of storage glycoproteins in mature pea (Pisum sativum) seeds by ion-spray tandem mass spectrometry (IS-MS/MS). Biosci. Biotechnol. Biochem., 60, 1841–1850.[ISI][Medline]

Kondo, A., Suzuki, J., Kuraya, N., Hase, S., Kato, I., and Ikenaka, T. (1990) Improved method for fluorescence labeling of sugar chains with sialic acid residues. Agric. Biol. Chem., 54, 2169–2170.[ISI][Medline]

Kornfeld, R. and Kornfeld, S. (1985) Assemby of asparagine-linked oligosaccharides. Annu. Rev. Biochem., 54, 631–664.[CrossRef][ISI][Medline]

Kukuruzinski, M.A. and Lennon, K. (1998) Protein N-glycosylation: molecular genetics and functional significance. Crit. Rev. Oral Biol. Med., 9, 415–448.[Abstract]

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[ISI][Medline]

Leiter, H., Mucha, J., Staudacher, E., Grimm, R., Glössl, J., and Altmann, F. (1999) Purification, cDNA cloning, and expression of GDP-L-Fuc: Asn-linked GlcNAc {alpha}1,3-fucosyltransferase from mung beans. J. Biol. Chem., 274, 21830–21839.[Abstract/Free Full Text]

Lerouge, P., Cabanes-Macheteau, M., Rayon, C., Fischette-Laine, A.C., Gomord, V., and Faye, L. (1998) N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol. Biol., 38, 31–48.[CrossRef][ISI][Medline]

Magnuson, N.S., Linzmaier, P.M., Reeves, R., An, G., HayGlass, K., and Lee, J.M. (1998) Secretion of biologically active human interleukin-2 and interleukin-4 from genetically modified tobacco cells in suspension culture. Protein Exp. Purif., 13, 45–52.[CrossRef][ISI][Medline]

Matsumoto, S., Ikura, K., Ueda, M., and Sasaki, R. (1995) Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells. Plant Mol. Biol., 27, 1163–1172.[ISI][Medline]

Misaki, R., Kimura, Y., Fujiyama, K., and Seki, T. (2001) Glycoproteins secreted from suspension-cultured tobacco BY2 cells have distinct glycan structures from intracellular glycoproteins. Biosci. Biotechnol. Biochem., 65, 2482–2488.[CrossRef][ISI][Medline]

Nagata, T., Okada, K., Takebe, I., and Matsui, C. (1981) Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (liposome). Mol. Gen. Cenet., 184, 161–165.

Ogawa, H., Hijikata, A., Amano, M., Kojima, K., Ishizuka, I., Kurihara, Y., and Matsumoto, I. (1996) Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergen Cry jI: relationship between the structures and antigenic epitopes of plant N-linked complex-type glycans. Glycoconj. J., 13, 555–566.[ISI][Medline]

Palacpac, N.Q., Yoshida, S., Sakai, H., Kimura, Y., Fujiyama, K., Yoshida, T., and Seki, T. (1999a) Stable expression of human ß1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns. Proc. Natl Acad. Sci. USA, 96, 4692–4697.[Abstract/Free Full Text]

Palacpac, N.Q., Kimura, Y., Fujiyama, K., Yoshida, T., and Seki, T. (1999b) Structures of N-linked oligosaccharides of glycoproteins from tobacco BY2 suspension cultured cells. Biosci. Biotechnol. Biochem., 63, 35–39.[ISI][Medline]

Rademacher, T.W., Parekh, R.B., and Dwek, R.A. (1988) Glycobiology. Ann. Rev. Biochem., 57, 785–838.[CrossRef][ISI][Medline]

Rayon, C., Lerouge, P., and Faye, L. (1998) The protein N-glycosylation in plants. J. Exp. Bot., 49, 1463–1472.[Abstract]

Ree, R., Cabanes-Macheteau, M., Akkerdaas, J., Milazzo, J.P., Loutelier-Bourhis, C., Rayon, C., Villalba, M., Koppelman, S., Aalberse, R., Rodriguez, R., and others. (2000) ß(1,2)-Xylose and {alpha}(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J. Biol. Chem., 275, 11451–11458.[Abstract/Free Full Text]

Richter, L.J., Thanavala, Y., Arntzen, C.J., and Mason, H.S. (2000) Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nature Biotechnol., 18, 1167–1171.[CrossRef][ISI][Medline]

Schauer, R. (1985) Sialic acids and their roles as biological masks. Trends Biochem. Sci., 10, 357–360.[CrossRef][ISI]

Shimazaki, A., Makino, Y., Omichi, K., Odani, S., and Hase, S. (1999) A new sugar chain of the proteinase inhibitor from latex of Carica papaya. J. Biochem., 125, 560–565.[Abstract]

Takahashi, N., Hotta, T., Ishihara, H., Mori, M., Tejima, S., Bligny, R., Akazawa, T., Endo, S., and Arata, Y. (1986) Xylose-containing common structural unit in N-linked oligosaccharides of laccase from sycamore cells. Biochemistry, 25, 388–395.[ISI]

Terashima, M., Murai, Y., Kawamura, K., Nakanishi, S., Stoltz, T., Chen, L., Drohan, W., Rodriguez, R.L., and Katoh, S. (1999) Production of functional human {alpha}1-antitrypsin by plant cell culture. Appl. Microbal. Biotechnol., 52, 523–526.

Thomas, H.T., Stephen, J.R., Yupin, C., Stephen, L.H., Victoria, F., and Laurence, K.G. (1995) Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Bio/Technology, 13, 53–57.[ISI][Medline]

Tomiya, N. and Takahashi, N. (1998) Contribution of component monosaccharides to the coordinates of neural and sialyl pyridylaminated N-glycans on a two-dimensional sugar map. Anal. Biochem., 264, 204–210.[CrossRef][ISI][Medline]

Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology, 3, 97–130.[Abstract]

Wilson, I.B.H., Zeleny, R., Kolarich, D., Staudacher, E., Stroop, C.J.M., Kamerling, J.P., and Altmann, F. (2001) Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core {alpha}1,3-linked fucose and xylose substitutions. Glycobiology, 11, 261–274.[Abstract/Free Full Text]

Yang, B.Y., Gray, J.S.S., and Montgomery, R. (1996) The glycans of horseradish peroxidase. Carbohydr. Res., 287, 203–212.[CrossRef][ISI][Medline]

Yusibov, V., Modelska, A., Steplewski, K., Agadjanyan, M., Weiner, D., Hooper, D.C., and Koprowski, H. (1997) Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1. Proc. Natl Acad. Sci. USA, 94, 5784–5788.[Abstract/Free Full Text]

Zeng, Y., Bannon, G., Thomas, V.H., Rice, K., Drake, R., and Elbein, A. (1997) Purification and specificity of ß1,2-xylosyltransferse, an enzyme that contributes to the allergenicity of some plant proteins. J. Biol. Chem., 272, 31340–31347.[Abstract/Free Full Text]





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
13/3/199    most recent
cwg021v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (5)
Disclaimer
Request Permissions
Google Scholar
Articles by Misaki, R.
Articles by Seki, T.
PubMed
PubMed Citation
Articles by Misaki, R.
Articles by Seki, T.