Remodeling of sugar chain structures of human interferon-{gamma}

Kazuhiro Fukuta, Reiko Abe, Tomoko Yokomatsu, Naoko Kono, Mineko Asanagi, Fumio Omae, Mari Toba Minowa2, Makoto Takeuchi2 and Tadashi Makino1

Life Science Laboratory, Mitsui Chemicals Inc., 1144 Togo, Mobara, Chiba 297–0017, Japan and 2Central Laboratories for Key Technology, KIRIN Brewery Co., Ltd., 1–13–5 Fuku-ura, Kanazawa-ku, Yokohama 236–0004, Japan

Received on September 20, 1999; revised on November 11, 1999; accepted on November 11, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Natural human interferon (IFN)-{gamma} has mainly biantennary complex-type sugar chains and scarcely has multiantennary structures. We attempted to remodel the sugar chain structures using IFN-{gamma} as a model glycoprotein. To obtain the branching glycoforms of IFN-{gamma}, we introduced the genes for GnT-IV (UDP-N-acetylglucosamine:{alpha}-1,3-D-mannoside ß-1,4-N-acetylglucosaminyltransferase) and/or GnT-V (UDP-N-acetylglucosamine:{alpha}-1,6-D-mannoside ß-1,6-N-acetylglucosaminyltransferase) into Chinese hamster ovary (CHO) cells producing human IFN-{gamma}. The parental CHO cells produced IFN-{gamma} with biantennary sugar chains mainly. When the GnT-IV activity was increased, triantennary sugar chains with a branch produced by GnT-IV increased up to 66.9% of the total sugar chains. When the GnT-V activity was increased, triantennary sugar chains with a corresponding branch increased up to 55.7% of the total sugar chains. When the GnT-IV and -V activities were increased at a time, tetraantennary sugar chains increased up to 56.2% of the total sugar chains. The proportion of these multiantennary sugar chains corresponded to the intracellular activities of GnT-IV and -V. What is more, lectin blot and flow cytometric analysis indicated that the multi-branch structure of the sugar chains was increased not only on IFN-{gamma}, one of the secretory glycoproteins, but also on almost CHO cellular proteins by introducing either or both of the GnT genes. The results suggest that the branching structure of sugar chains of glycoproteins could be controlled by cellular GnT-IV and GnT-V activities. This technology can produce glycoforms out of natural occurrence, which should enlarge the potency of glycoprotein therapeutics.

Key words: GnT-IV/GnT-V/interferon-{gamma}/N-glycosylation/remodeling


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Glycosylation is a significant posttranslational modification of most proteins, and can play important roles in defining the properties of glycoproteins, including biological activity, immunogenicity, pharmacokinetics, solubility, and protease resistance (reviewed in Kobata, 1992Go; Varki, 1993Go). Recently, various glycosyltransferase genes have been cloned (reviewed in Field and Wainwright, 1995Go; Taniguchi and Ihara, 1995Go), making it possible to change the sugar chain structure of a glycoprotein by introducing glycosyltransferase genes into host cells. We have been attempting to improve the function of glycoproteins by remodeling their sugar chains, with the aim of producing industrially useful glycoproteins with improved functions.

For controlling and remodeling of the sugar chain structure, control of the branching structure as the skeletal structure is a key step. It is known that the highly branched sugar chains are essential for the effective expression of in vivo biological activity of erythropoietin (Takeuchi et al., 1989Go). The branching structure of the complex-type sugar chain is controlled by various N-acetylglucosaminyltransferases (GnTs). These enzymes are classified as I to VI (Schachter et al., 1983Go; Schachter, 1986Go), and work in the middle stage of the sugar chain biosynthesis, being responsible for the formation of the skeletal structure of sugar chains. The GnTs involved in the formation of multiantennary sugar chains are GnT-IV (UDP-N-acetylglucosamine:{alpha}-1,3-D-mannoside ß-1,4-N-acetylglucosaminyltransferase, EC 2.4.1.145) and GnT-V (UDP-N-acetylglucosamine:{alpha}-1,6-D-mannoside ß-1,6-N-acetylglucosaminyltransferase, EC 2.4.1.155) (Figure 1, Table I). GnT-V cDNA was isolated from mammalian cells several years ago (Shoreibah et al., 1993Go; Saito et al., 1994Go), however GnT-IV cDNA had not been cloned until recently. With the success in the cDNA cloning of GnT-IV achieved by Minowa et al. (1998)Go, it is now possible to study the effect on sugar chain structures caused by the overexpression of these enzymes.



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Fig. 1. Reaction pathway of GnT-IV and GnT-V.

 


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Table I. Structures and abbreviations of PA-sugar chains

 
In the present study, we attempt to remodel the sugar chain structures of human IFN-{gamma}. IFN-{gamma} was considered an appropriate model because its N-linked sugar chain structure has been well studied (reviewed in Hooker and James, 1998Go). Moreover, IFN-{gamma} has potential as a therapeutic drug in the treatment of certain types of cancer, in addition to antiviral activity. Human IFN-{gamma} molecule contains two potential N-glycosylation sites at residues 25 and 97 (Rinderknecht et al., 1984Go; Sareneva et al., 1996Go). In gel electrophoresis, three monomeric forms of IFN-{gamma} are found with apparent molecular weights of 25,000, 20,000, and 17,000, corresponding to molecules with two, one or none of the glycosylation sites occupied. Sugar chains of natural human IFN-{gamma} from peripheral-blood lymphocytes or myelomonocytes and recombinant human IFN-{gamma} from Chinese hamster ovary (CHO) cells are mainly biantennary structures with microheterogeneity as to the core fucose residue and terminal N-acetylneuraminic acids (Mutsaers et al., 1986Go; Yamamoto et al., 1989aGo,b).

In this report, we demonstrate the remodeling of biantennary sugar chains of IFN-{gamma} into multiantennary structures. We introduce the genes encoding GnT-IV and/or GnT-V into CHO cells producing human IFN-{gamma} and investigate the sugar chain structures of the IFN-{gamma} produced. In addition, we describe the relationship between the changes in the sugar chain structures of IFN-{gamma} secreted from cells and those of cellular proteins.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Preparation of cell lines overexpressing GnT-IV and/or GnT-V
IM4 clone, a human IFN-{gamma} producing CHO cell line, was transfected with GnT-IV expression vector pCXN2-bGnT-IV (containing neomycin-resistance gene) or GnT-V expression vector pCXH1-hGnT-V (containing hygromycine-resistance gene). Thirty G418-resistant clones and 50 hygromycine-resistant clones were isolated from cells transfected with GnT-IV gene and GnT-V gene, respectively. IM4/neo and IM4/hygro were obtained as negative transfectants by introducing pCXN2 and pCXH1, respectively. Clones overexpressing the introduced gene with no difference in ß-1,4-GalT activity and IFN-{gamma} productivity compared to the parental IM4 clone were selected.

Among the G418-resistant clones, one clone designated as IM4/IV was selected as a GnT-IV high expression clone. Among the hygromycine-resistant clones, two clones designated as IM4/Vh and IM4/Vm were selected according to their high and moderate expression of GnT-V, respectively. Activities of GnT-IV and GnT-V in each clone are summarized in Table II. While the GnT-IV activity was not detected in parental cells, IM4/IV clone had an extraordinary high activity of GnT-IV. The GnT-V activities of IM4/Vh and IM4/Vm were approximately 17-fold and 4-fold higher than that of parental cells, respectively.


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Table II. Glycosyltransferase activities (nmol/h/106 cells) in parental CHO cells and transfectants
 
IM4/Vh clone was further transfected with GnT-IV expression vector pCXN2-bGnT-IV and 30 clones with both hygromycine- and G418-resistance were isolated. IM4/V/neo clone was obtained as a negative control (vector pCXN2 was introduced). One clone with high GnT-IV activity, in which IFN-{gamma} productivity and the activities of GnT-V and ß-1,4-GalT were of the same level as in IM4/Vh, was selected as a GnT-IV/-V high expression clone and designated as IM4/V/IV.

Sugar chain structures of IFN-{gamma} produced by the parental CHO clone and GnT-IV and/or GnT-V transfectants
Sugar chains at two glycosylation sites of IFN-{gamma} produced by each CHO clone were collectively excised and purified, and the structures were analyzed by the two-dimensional mapping technique of Tomiya et al. (1988)Go using pyridylaminated (PA-) sugar chains (Table I). Reversed phase HPLC profiles of desialylated PA-sugar chains derived from IFN-{gamma} produced by CHO cells are shown in Figure 2. The proportion of each sugar chain is shown in Table III.



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Fig. 2. Reversed phase HPLC chromatograms of desialylated PA-sugar chains derived from IFN-{gamma} produced by parental CHO cells and transfectants. PA-sugar chains from each IFN-{gamma} were desialylated and analyzed by reversed phase HPLC. Each peak was further separated by normal phase HPLC and the structure was identified. Peaks that disappeared or were reduced by endo-ß-galactosidase digestion are indicated by asterisks. Arrows indicate the elution positions of authentic PA-sugar chains listed in Table I.

 


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Table III. Structures of desialylated sugar chains of IFN-{gamma} produced^Mby parental CHO cells and transfectants

n.d., Not detected.

aPercentage is given as a total of other forms.

 
Regarding the IFN-{gamma} sugar chain composition in the parental clone IM4, the majority consisted of biantennary sugar chains, comprising 66.1% (total amount of A, B, C, D, E, and F). Sugar chains of IFN-{gamma} produced by the GnT-IV high expression clone IM4/IV were mainly triantennary structures with GlcNAcß1–4 branch on the Man{alpha}1–3 arm of trimannosylcore, comprising 63.9% (total amount of H and I). Sugar chain structures of IFN-{gamma} produced by the GnT-V high expression clone IM4/Vh were mainly triantennary structures with GlcNAcß1–6 branch on the Man{alpha}1–6 arm of trimannosylcore, comprising 28.9% (total amount of K and L) of the overall sugar chains. In IFN-{gamma} from IM4/Vh clone, there were also some components other than those shown in Table III. They comprised over 50%. Since most of their HPLC peaks (Figure 2, indicated by asterisks) disappeared or were reduced by digestion with endo-ß-galactosidase, they were assumed to be triantennary structures with N-acetyllactosamine repeats, although individual structures were not determined. In IFN-{gamma} from the GnT-V moderate expression clone IM4/Vm, triantennary structures with GlcNAcß1–6 branch on the Man{alpha}1–6 arm of trimannosylcore were increased (K and L) compared to in IFN-{gamma} from parental clone IM4. The extent of the increase was as low as 14.9% of all sugar structures, reflecting the degree of GnT-V activity elevation. In IFN-{gamma} from IM4/V/IV clone highly expressing both GnT-IV and GnT-V, tetraantennary sugar structures (N and O) were major, comprising 34.5% of the entire sugar chains. There were also some structures other than those shown in Table III as in the GnT-V high expression clone and they comprised nearly 50%. Most of their HPLC peaks (Figure 2, indicated by asterisks) disappeared or were reduced by digestion with endo-ß-galactosidase, suggesting the presence of N-acetyllactosamine repeats.

Analysis of the branching degree of sugar chains
As described above, in IFN-{gamma} from IM4/Vh clone and IM4/V/IV clone, there seemed to be more sugar chains with the multiantennary backbone structures in addition to those shown in Table III. They were considered to have N-acetyllactosamine repeats. Therefore, to estimate the branching degrees of sugar chains precisely, the desialylated PA-sugar chain mixture excised from each IFN-{gamma} was digested with ß-galactosidase (jack bean), endo-ß-galactosidase (Escherichia freundii) and {alpha}-fucosidase (bovine kidney) simultaneously to convert each PA-sugar chain to its backbone structure. Then the backbone sugar chain mixture obtained was separated and quantitated by HPLC. Figure 3 and Table IV show the reversed phase HPLC chromatograms and results of quantitation, respectively. Differences in the amounts of the triantennary chain having GlcNAcß1–6 branch and the tetraantennary chain were observed between the values in Table IV and those calculated from Table III, meaning that N-acetyllactosamine repeats were formed on these chains. It was revealed that sugar chains containing triantennary structure with the GlcNAcß1–6 branch as the backbone structure comprised 55.7% in IM4/Vh clone. In the IM4/V/IV clone, sugar chains containing tetraantennary structures as the backbone structure comprised 56.2%. The amount of each backbone structure was similar to that calculated from Table III in IM4/IV clone. Thus, analysis of the sugar chain backbone structures clearly showed the branching degree of sugar chains in each IFN-{gamma}. From this analysis, it was revealed that more than half of the sugar chains linked to IFN-{gamma} were converted to the multiantennary types in the cells transfected with GnT-IV and/or GnT-V genes.



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Fig. 3. Reversed phase HPLC chromatograms of skeletal PA-sugar chains derived from IFN-{gamma} produced by parental CHO cells and transfectants. PA-sugar chains from each IFN-{gamma} were converted to the skeletal structures by digestion with the glycosidase mixture and analyzed by reversed phase HPLC. The structure of each peak is indicated by an alphabetical symbol shown in Table I. Fractions indicated by bars were collected and further separated under different conditions as described in Materials and methods. UnK1, UnK2, and UnK3 are the peaks of which structures could not be identified.

 


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Table IV. Skeletal sugar chain structures of IFN-{gamma} produced by parental CHO cells and transfectants

PA-sugar chains from each IFN-{gamma} were converted to the skeletal structures by digestion with a glycosidase mixture and quantitated as described in Materials and methods. The structures of UnK1, UnK2, and UnK3 were not identified. n.d., not detected.

aPercentage is given as a total of minor peaks. Each peak area was not more than 1% of the total area.

 
In this analysis, it was also found that skeletal sugar chain structures other than G0-Bi-PA, G0-Tri-PA, G0-Tri'-PA, and G0-Tetra-PA, designated as UnK-1, UnK-2, and UnK-3, were present. However, the amounts of these sugar chains were too small to determine the precise structures, molecular masses were determined by MALDI-TOF/MS analysis to deduce the structures. Based on the molecular masses, UnK1, UnK2, and UnK3 were assumed to be HexNAc4Hex3 (detected (M+Na)+ mass, 1415.4; calculated (M+Na)+ mass, 1418.3), HexNAc5Hex3 (detected (M+Na)+ mass, 1620.3; calculated (M+Na)+ mass, 1621.5) and HexNAc6Hex3 (detected (M+Na)+ mass, 1824.1; calculated (M+Na)+ mass, 1824.7), respectively, in which an increase in molecular mass corresponding to one molecule of HexNAc was detected (data not shown).

The rate of sialylation
PA-sugar chains from IFN-{gamma} produced by each CHO cell line were analyzed by anion exchange HPLC and the rate of sialic acid addition to IFN-{gamma} sugar chains was measured (Figure 4). There was no clear tendency such as increase in the average number of sialic acids along with increase in the branching degree of sugar chains. However, some differences were observed in the proportion of sugar chains having different numbers of sialic acids among IFN-{gamma} from IM4, IM4/Vm, IM4/Vh, and IM4/V/IV clones. In IM4/Vm, IM4/Vh, and IM4/V/IV clones, mono and disialo sugar chains decreased, while trisialo and tetrasialo sugar chains increased. These results were interpreted as a reflection of the increase in the branches of sugar chains. In IM4/V/IV clone, asialo sugar chains also increased. The reason for this is unclear. The overall proportion in IM4/IV was similar to that in IM4.



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Fig. 4. Analysis of sialylation. (A) Anion exchange HPLC chromatograms of PA-sugar chains derived from IFN-{gamma} produced by parental CHO cells and transfectants. PA-sugar chains from each IFN-{gamma} were separated according to the negative charges. Arrows indicate the elution positions of: 0, asialo PA-sugar chains; 1, monosialo PA-sugar chains; 2, disialo PA-sugar chains; 3, trisialo PA-sugar chains; 4, tetrasialo PA-sugar chains. The rate of sialic acid addition in each IFN-{gamma} is shown in (B).

 
Analysis of the sugar chain structure of cellular proteins in each clone
Cell lysates of the parental clone IM4 and GnT-IV and/or GnT-V transfectants were analyzed by lectin blot analysis using DSA (Datura stramonium agglutinin) lectin, which recognizes multiantennary sugar chains. The blot was stained by DSA after treating with endo-ß-galactosidase in order to exclude the reaction of DSA with the polylactosamine (it is also recognized by DSA). While IM4 cell lysates showed almost no reactivity for DSA, all cell lysates from the GnT-IV and/or GnT-V transfectants reacted with DSA (Figure 5). This finding indicated that sugar chains of most cellular proteins were changed to multiantennary structures by the overexpression of GnT-IV and/or GnT-V.



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Fig. 5. Lectin blot analysis of whole proteins from the parental CHO cells and transfectants. Cell lysates were separated by SDS–PAGE and transferred onto a PVDF membrane. The blot was probed with DSA after treating with sialidase and endo-ß-galactosidase. The positions of molecular size markers are indicated on the right.

 
Flow cytometric analysis was also performed using DSA lectin to analyze sugar chain structures on the cell surfaces. DSA-bound sugar chains increased on the cell surfaces of the GnT-IV and/or GnT-V transfectants compared to the cell surface of IM4 clone (Figure 6). The binding capacity to DSA was higher on the surface of GnT-V high expression clone IM4/Vh than on that of GnT-V moderate expression clone IM4/Vm, reflecting the degree of GnT-V activity elevation. It was shown that the cell surface sugar chains were converted to multiantennary types by the overexpression of GnT-IV and/or GnT-V.



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Fig. 6. Flow cytometric analysis of cell surfaces of the parental CHO cells and transfectants. (A) Flow cytometry histograms. Cells were incubated with FITC-DSA and analyzed by flow cytometer. The black histogram indicates fluorescence of stained cells, and the white histogram indicates autofluorescence of the cells. Fluorescence intensity is shown as a log scale. (B) The binding capacity with DSA was evaluated by the difference between the mean fluorescence of stained cells and the mean autofluorescence.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
It is known that sugar chains of natural human IFN-{gamma} have complex-type biantennary structures with microheterogeneity as to the core fucose residue and terminal N-acetylneuraminic acids (Yamamoto et al., 1989aGo,b). Recombinant human IFN-{gamma} produced by CHO cells has also complex-type biantennary sugar chains with similar microheterogeneity although the linkage (only {alpha}-2,3 linkage) of N-acetylneuraminic acids is different from natural human IFN-{gamma} (containing both {alpha}-2,3 and {alpha}-2,6 linkages) (Mutsaers et al., 1986Go). Thus, biantennary sugar chains are commonly linked to IFN-{gamma}. The reason for this was considered that GnT-IV and GnT-V are hardly accessible to the sugar chains of IFN-{gamma}, or the expression level of GnT-IV and GnT-V in host cells is low. Since it has been known that only a small percentage of glycoproteins contains the GlcNAcß1–6 branch, the GnT-V product, it has been generally considered that whether the highly branched complex-type sugar chain is formed or not is largely dependent on the protein backbone structure (Do and Cummings, 1993Go; Do et al., 1994Go).

In contrast, we verified that the sugar chain structures of IFN-{gamma} are changeable to multiantennary type, by increasing the GnT-IV and/or GnT-V expression level in IFN-{gamma} producing cells. The proportion of multiantennary sugar chains corresponded to the intracellular activities of these enzymes. This explains why biantennary sugar chains are commonly attached to IFN-{gamma}: not because GnT-IV or GnT-V is hardly accessible to the sugar chains of IFN-{gamma}, but because of the low intracellular expression level of GnT-IV and GnT-V.

It was also found by lectin blot and flow cytometric analysis that not only did the sugar chains of secretory IFN-{gamma} change to the multiantennary structure, but so did most of the sugar chains of the cellular proteins of the CHO cells transfected with GnT-IV or GnT-V. Thus, there was a close relationship between the sugar chain structures of IFN-{gamma}, which is secreted from the cells, and those of other cellular proteins. This finding shows a great influence of the glycosyltransferase expression level in the host cells on the structures of the sugar chains. The present study has demonstrated that the sugar chains of a glyco­protein can be mostly modified by changing the glyco­syltransferase activities in the host cells.

In the analysis of the skeletal structures of the sugar chains in IFN-{gamma}, structures other than G0-Bi-PA, G0-Tri-PA, G0-Tri'-PA, and G0-Tetra-PA, designated as UnK-1, UnK-2, and UnK-3, were found. Although the precise structures of these UnK sugar chains could not be defined, UnK1, UnK2, and UnK3 were assumed to be HexNAc4Hex3-PA, HexNAc5Hex3-PA, and HexNAc6Hex3-PA, respectively, in which an increase in molecular mass corresponding to one molecule of HexNAc was detected in MALDI-TOF/MS analysis. From these results and the finding that UnK1, UnK2, and UnK3 were mainly present in IFN-{gamma} from parental clone, GnT-V transfectant, and GnT-IV/-V transfectant, respectively, it was estimated that UnK1 was converted to UnK2 by the GnT-V reaction, and that UnK2 was further converted to UnK3 by the GnT-IV reaction. Considering the change of these UnK sugar chains, it was concluded that most sugar chains of IFN-{gamma} have been changed by the action of GnT-IV and/or GnT-V introduced to the host cells.

As shown above, we have demonstrated that the biantennary structure of N-glycans can be mostly remodeled to highly branched structures by increasing the intracellular activities of GnT-IV and/or GnT-V, contrary to what was previously believed. Although normal cells do not have such high activities of GnT-IV and GnT-V, which is also the case with CHO cells, it is obvious that overexpression of these enzymes can provide highly branched N-glycans. This finding enables us to produce highly branched glycoforms out of natural occurrence. Therefore, GnT-IV and GnT-V are considered useful tools for investigating the potency of remodeling of sugar chains to improve functions of glycoproteins.

Structural analyses of sugar chains also revealed that the poly-N-acetyllactosamine structures increased in IFN-{gamma} produced by GnT-V high expression clone and GnT-IV/-V high expression clone, reflecting the substrate specificity of iGnT (UDP-N-acetylglucosamine:N-acetyllactosamide ß1–3-N-acetylglucosaminyltransferase) for GlcNAcß1–6 branch (Van den Eijnden et al., 1988Go). These results were consistent with previous reports (Yousefi et al., 1991Go; Do and Cummings, 1993Go).

In the analysis of sialylation, slight increases in the sialic acid addition were observed in IM4/Vm, IM4/Vh, and IM4/V/IV clone. However, the increases were not so significant as was expected from the increase in the branching degree of the sugar chains in IFN-{gamma} produced by GnT-IV and/or GnT-V transfectants. This was considered due to insufficient activities of sialyltransferase and/or CMP-sialic acid transporter in CHO cells. Since the rate of sialylation is known to influence the pharmacokinetics, we are trying to augment sialylation in the IFN-{gamma} of which sugar chains were remodeled to multiantennary structures.

It is reported that the sugar chains of IFN-{gamma} influence the antiviral activity and pharmacokinetics in addition to physical properties such as protease resistance (Arakawa et al., 1986Go; Bocci et al., 1985Go; Sareneva et al., 1993Go, 1994, 1995). It is therefore of interest to elucidate the properties imparted by each of the sugar chain structures of IFN-{gamma}. The work is in progress.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
GENETICIN (G418), CHO-S-SFM II medium, CD-CHO medium, and OPTI-MEM I medium were purchased from Life Technologies, Inc.. Hygromycine B was from Wako Pure chemical Industries, Japan. Methotrexate was from Sigma Chemical Company. Dialyzed fetal bovine serum (dFBS) was from JRH Biosciences. Glycopeptidase A (Almond), ß-galactosidase (jack bean), endo-ß-galactosidase (Escherichia freundii ), ß-N-acetylhexosaminidase (jack bean), biotinylated DSA, and FITC-DSA were obtained from Seikagaku Kogyo, Japan. Sialidase (neuraminidase, from Arthrobacter ureafaciens ) was from Nacalai tesque, Japan. {alpha}-fucosidase (bovine kidney) was from Oxford Glycoscience, UK. PA-Sugar chain standards were from Takara Shuzo Co., Japan.

Culture of CHO cells
CHO cells producing human IFN-{gamma} were routinely maintained by static culture at 37°C (5% CO2/air) in tissue culture flasks containing CHO-S-SFM II medium and 10% dialyzed fetal bovine serum (dFBS) supplemented with 250 nM methotrexate (MTX). G418 (300 µg/ml) and hygromycine B (200 µg/ml) were added to GnT-IV and GnT-V transfected cell cultures, respectively.

For IFN-{gamma} production, proliferated cells were cultured in triple flasks (500 cm2) containing 200 ml of the medium for 4 days. Then the cells were washed with PBS and the medium was replaced by 200 ml of serum-free CD-CHO medium supplemented with 2 mM L-glutamine. The medium was replaced with 200 ml of fresh medium at 24 h intervals and culture super­natants were filtered (0.22 µm) and stored at –20°C.

Establishment of a cell line with high IFN-{gamma} productivity
HIIF-D cell line producing human IFN-{gamma} (obtained from ATCC, No. CRL-8200) was established from CHO cells deficient in dihydrofolate reductase (DHFR-) by cotransfection of pSV-IFN-{gamma} (containing the coding sequence for human IFN-{gamma}) and pAdD26SV(A)-3 (containing the coding sequence for the mouse DHFR) (Scahill et al., 1983Go). HIIF-D subclone stably producing a high level of IFN-{gamma}, designated as IM4, was used in the following experiments.

Construction of the GnT-IV and GnT-V expression vectors
GnT-IV expression vector pCXN2-bGnT-IV was constructed by inserting the entire bovine GnT-IV coding region (XhoI/SmaI fragment of pBGT4, blunt ended) (Minowa et al., 1998Go) into the EcoRI site (blunt ended) of an expression vector pCXN2 (kindly provided by Dr. Jun-ichi Miyazaki, Osaka University), which is regulated by ß-actin promoter and contains neomycin-resistance gene (Niwa et al., 1991Go). Human GnT-V cDNA was obtained from a human fetal brain-derived cDNA library (CLONTECH) by PCR on the basis of the nucleotide sequence reported (Shoreibah et al., 1993Go; Saito et al., 1994Go). Primer sequences for PCR were 5'-CTTCTCGAGGTTAAGAGCCA­AGGACAGGTGAAGTTGCCA-3' and 5'-AGGCTCGAGCT­ATAGGCAGTCTTTGCAGAGAGCC-3'. GnT-V expression vector pCXH1-hGnT-V was constructed by inserting the entire human GnT-V coding region (XhoI fragment) into the XhoI site of an expression vector pCXH1, which is regulated by ß-actin promoter and contains hygromycin-resistance gene. pCXH1 was constructed by inserting the SalI/HindIII fragment of pCXN2 encoding chicken ß-actin promoter into the HindIII/XhoI site of pBS-Hyg-NN. pBS-Hyg-NN was constructed by inserting the NarI/AccI fragment encoding the hygromycine-resistance gene of pCEP4 (Invitrogen), which was blunt-ended followed by the addition of NotI linker, into the NotI site of pBlueScriptII-KS+ (Strategene).

Transfection of GnT-IV and GnT-V genes
Forty micrograms of plasmid and 4 x 106 cells were suspended in 0.4 ml of OPTI-MEM I medium and subjected to electro­poration (220 V/0.4 cm, 960 µ F). After incubating at room temperature for 10 min, the cells were added to CHO-S-SFMII medium (supplemented with 10% dFBS and 250 nM MTX) and seeded on plates at an appropriate dilution. Two days later, selection marker compounds (300 µg/ml G418 for GnT-IV and 200 µg/ml hygromycine B for GnT-V) were added and the cells were further cultured. After approximately two weeks, drug-resistant cells forming colonies were picked up.

Assay of glycosyltransferase activities
CHO cells (5 x 105) were suspended in 5 µl of buffer (10 mM HEPES buffer, 1% Triton X-100, pH 7.2) and disrupted by sonication. The lysates were used as the crude enzyme preparations for the assay of glycosyltransferase activities.

Activities of GnT-IV and GnT-V were measured using pyridylaminated agalacto biantennary sugar chain (G0-Bi-PA, shown in Table I) as a substrate by the method of Nishikawa et al. (1990)Go with some modifications. Preparation of the substrate was done as reported (Tokugawa et al., 1996Go). The reaction mixture for GnT-IV contained 10 mM HEPES buffer (pH 7.2), 80 mM UDP-GlcNAc, 10 mM MnCl2, 33 mM NaCl, 3 mM KCl, 200 mM GlcNAc, 0.2% (W/V) Triton X-100, and 800 µM pyridylaminated substrate in a total volume of 25 µl. The reaction mixture for GnT-V was the same as that for GnT-IV except addition of 10 mM EDTA instead of 10 mM MnCl2. The reaction was performed at 37°C for 2 h, then 4 µl of the reaction mixture was taken for analysis and added to 96 µl of stop solution (10 mM HEPES, 50 mM EDTA, pH 7.2). The reaction was stopped by boiling for 3 min. The reaction products were analyzed by reversed phase HPLC using a Shim-pack CLC-ODS column (6 x 150 mm, Shimadzu, Japan). The specific activities of the enzymes were expressed as nmol of GlcNAc transferred per hour per 106 cells. GalT activity was assayed by the same method except that the reaction mixture contained 10 mM HEPES buffer (pH 7.2), 80 mM UDP-Gal, 10 mM MnCl2, 33 mM NaCl, 3 mM KCl, 5.6 mM {gamma}-galactonolactone, 0.2% (W/V) Triton X-100, and 800 µM pyridylaminated substrate in a total volume of 25 µl. The specific activity of the enzyme was expressed as nmol of galactose transferred per hour per 106 cells.

Purification of IFN-{gamma}
IFN-{gamma} was purified by immunoaffinity chromatography. The immunoaffinity column was prepared by coupling polyclonal anti-human IFN-{gamma} antibody (PIF-3, Hayashibara, Japan) to NHS-activated Sepharose HP (Amersham Pharmacia Biotech). Cell culture supernatant was loaded onto the column equilibrated with 50 mM Tris–HCl and 0.5 M NaCl, pH 7.5. After washing with CHROMATOP WASH BUFFER A (Nihon-gaishi, Japan), IFN-{gamma} was eluted with 0.2 M Gly-HCl, pH 2.5. The eluate was immediately neutralized with 1 M Tris–HCl, pH 8.0 and concentrated and desalted by ultrafiltration with Ultrafree 15 centrifugal filter (Millipore).

Release of sugar chains from IFN-{gamma} and pyridylamination
Purified IFN-{gamma} was redissolved in 200 µl of 6 M urea and incubated at 60°C for 1 h, then digested with modified trypsin (Promega) at 37°C for 15 h in a final volume of 1.2 ml of 100 mM Tris–HCl, 1 mM CaCl2, and 1 M urea, pH 8.0. The digest was loaded on a Sephadex G-25 (Amersham Pharmacia Biotech) gel filtration column and glycopeptide fractions were collected. Glycopeptides were dissolved in 100 µl of 100 mM citrate-phosphate buffer, pH 5.0 and digested with 0.4 mU of glycopeptidase A at 37°C for 15 h. The digested products were applied to a Sep-Pak Plus C18 Cartridge (Waters) pretreated with 10 ml of methanol and 5 ml of water, then sugar chains were eluted with 6 ml of 5% acetonitrile in 0.1% trifluoroacetic acid solution.

The obtained sugar chains were pyridylaminated by the methods of Kuraya and Hase (1992)Go. To a lyophilized sample, 20 µl of 2-aminopyridine reagent (prepared by mixing 552 mg of 2-aminopyridine with 200 µl of acetic acid) was added and the mixture was heated at 90°C for 60 min. Next, 70 µl of a reducing reagent (prepared by mixing 200 mg of borane-dimethylamine complex, 50 µl of water, and 80 µl of acetic acid) was added and the mixture was heated at 80°C for 35 min. Excess reagents were removed by gel filtration on a Sephadex G-15 (Amersham Pharmacia Biotech) column (1 x 40 cm) equilibrated with 10 mM NH4HCO3.

Structural analysis of PA-sugar chains
The PA-sugar chains were first analyzed by anion-exchange HPLC and the content of sialic acid was determined. Next, the whole PA-sugar chains were desialylated with sialidase from Arthrobacter ureafaciens in 0.2 M ammonium acetate buffer, pH 5.0 for 20 h at 37°C. The desialylated sugar chains were separated as a neutral fraction on anion-exchange HPLC using a Mono Q column (5 x 50 mm, Amersham Pharmacia Biotech). Elution was performed at a flow rate of 1.0 ml/min at room temperature. The column was equilibrated with solvent A (H2O adjusted to pH 9.0 by aqueous ammonia) and the ratio of solvent B (1.0 M ammonium acetate, pH 9.0) to A was increased to A:B = 90:10 over 30 min by a linear gradient.

The desialylated PA-sugar chains were analyzed by a two-dimensional sugar mapping technique (Tomiya et al., 1988Go). First, the desialylated PA-sugar chains were separated by reversed phase HPLC on a Shim-pack CLC-ODS column (6 x 150 mm, Shimadzu, Japan). Each sugar chain fraction was collected separately and then applied to the second column, TSKgel Amide-80 (4.6 x 250 mm, Tosoh, Japan). Elution conditions for these columns were as described by Tomiya et al. (1988)Go. Each PA-sugar chain fraction was sequentially digested by exoglycosidases to verify the structural identification.

To analyze the branching structures of sugar chains, desialylated PA-sugar chains were simultaneously treated with ß-galactosidase (jack bean), endo-ß-galactosidase (Escherichia freundii), and {alpha}-fucosidase (bovine kidney). Obtained backbone sugar chains were separated and quantitated by reversed phase HPLC with Shim-pack CLC-ODS. Elution was firstly performed under the conditions described by Tomiya et al. (1988)Go. For the separation of G0-Bi-PA and G0-Tetra-PA, the fraction of these components was collected and eluted under different conditions: the elution was performed at 55°C with 10 mM phosphate buffer (pH 3.8) containing 0.025% 1-butanol at a flow rate of 1.0 ml/min.

In all HPLC systems, PA-sugar chains were detected by fluorescence with excitation and emission wavelengths of 320 nm and 400 nm, respectively.

MALDI-TOF/MS analysis was performed using KRATOS KOMPACT MALDI III (Shimadzu). Samples were prepared by mixing matrix solution: 10 mg/ml solution of 2,5-dihydroxybenzoic acid in 0.1% trifluoroacetic acid/ethanol (50/50, by volume).

Exo- and endoglycosidase digestion
Enzymatic digestion of PA-sugar chains was performed with the following enzymes at 37°C for 18 h. The PA-sugar chains were incubated with 20 mU {alpha}-fucosidase (bovine kidney) in 20 µl of 0.2 M ammonium acetate buffer, pH 4.5, with 20 mU ß-galactosidase (jack bean) in 20 µl of 0.1 M citrate-phosphate buffer, pH 4.0, or with 20 mU ß-N-acetylhexosaminidase (jack bean) in 20 µl of 0.1 M citrate-phosphate buffer, pH 5. The reaction mixture was heated at 100°C for 3 min to terminate digestion. For the removal of side chains of sugar chains in order to obtain the backbone structures, PA-sugar chains were simultaneously treated with 20 mU {alpha}-fucosidase (bovine kidney), 20 mU ß-galactosidase (jack bean), and 4 mU endo-ß-galactosidase (Escherichia freundii) in 100 µl of 0.1 M citrate-phosphate buffer, pH 5.0, at 37°C for 30 h.

Lectin blot analysis
Cells (1 x 106) were suspended in 0.3 ml of PBS and disrupted by sonication. The cell lysates were electrophoresed on a 4–20% SDS–polyacrylamide gradient gel by the method of Laemmli (Laemmli, 1970Go) and the gel was blotted onto a PVDF membrane (Immobilon-P, Millipore). After rinsing with Tris-buffered saline (TBS, pH 7.2), the membrane was blocked with 3% bovine serum albumin in TBS for 1 h. The membrane was rinsed with TBS and then desialylation was performed by incubation with sialidase (Arthrobacter ureafaciens) at 37°C for 15 h in 0.2 M ammonium acetate buffer, pH 5.0. The membrane was successively treated with endo-ß-galactosidase (Escherichia freundii) at 37°C for 15 h in 50 mM sodium acetate buffer, pH 5.8. After washing with TTBS (TBS containing 0.05% Tween 20), the membrane was incubated at room temperature with biotinylated DSA lectin in TTBS containing 1% BSA. The membrane was washed with TTBS and then incubated in horseradish peroxidase–avidin (VECTASTAIN ABC Kit, Vector) at room temperature for 1 h. The membrane was washed with TTBS and exposed to 3,3'-diaminobenzidine tetrahydrochloride.

Flow cytometric analysis
Confluent cells were removed from culture dishes using PBS, 0.25% trypsin, 0.025% EDTA and resuspended in PBS containing 0.1% BSA. FITC labeled DSA was added to a final concentration of 5 µg/ml. After standing for 30 min on ice, cells were washed with PBS containing 0.1% BSA, then analyzed by flow cytometer (EPICS ELITE, Coulter). Unstained cells were used as a control.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. Tamao Endo (Tokyo Metropolitan Institute of Gerontology, Japan) for advice on lectin blot analysis and helpful discussions. We also thank Dr. Jun-ichi Miyazaki (Osaka University, Japan) for the supply of vector pCXN2. This work was fully supported by New Energy and Industrial Technology Development Organization (NEDO) as a part of the Research and Development Projects of Industrial Science and Technology Frontier Program in Japan.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
IFN-{gamma}, interferon-{gamma}; GnT-IV, UDP-N-acetylglucosamine:{alpha}-1,3-D-mannoside ß-1,4-N-acetylglucosaminyltransferase; GnT-V, UDP-N-acetylglucosamine:{alpha}-1,6-D-mannoside ß-1,6-N-acetylglucosaminyltransferase; ß-1,4-GalT, UDP-galactose: N-acetylglucosamine ß-1,4-galactosyltransferase; CHO, Chinese hamster ovary; GlcNAc, N-acetyl-D-glucosamine; Man, D-mannose; Gal, D-galactose; Fuc, L-fucose; Hex, hexose; HexNAc, N-acetylhexosamine; PA, pyridylamino-; HPLC, high-performance liquid chromatography; DSA, Datura stramonium agglutinin; PBS, phosphate-buffered saline; chemical structures of sugar chains and their abbreviations are summarized in Table I.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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