Life Science Laboratory, Mitsui Chemicals Inc., 1144 Togo, Mobara, Chiba 2970017, Japan and 2Central Laboratories for Key Technology, KIRIN Brewery Co., Ltd., 1135 Fuku-ura, Kanazawa-ku, Yokohama 2360004, Japan
Received on September 20, 1999; revised on November 11, 1999; accepted on November 11, 1999.
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Abstract |
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Key words: GnT-IV/GnT-V/interferon-/N-glycosylation/remodeling
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Introduction |
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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., 1989). 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., 1983
; Schachter, 1986
), 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:
-1,3-D-mannoside ß-1,4-N-acetylglucosaminyltransferase, EC 2.4.1.145) and GnT-V (UDP-N-acetylglucosamine:
-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., 1993
; Saito et al., 1994
), 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)
, it is now possible to study the effect on sugar chain structures caused by the overexpression of these enzymes.
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In this report, we demonstrate the remodeling of biantennary sugar chains of IFN- into multiantennary structures. We introduce the genes encoding GnT-IV and/or GnT-V into CHO cells producing human IFN-
and investigate the sugar chain structures of the IFN-
produced. In addition, we describe the relationship between the changes in the sugar chain structures of IFN-
secreted from cells and those of cellular proteins.
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Results |
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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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Sugar chain structures of IFN- produced by the parental CHO clone and GnT-IV and/or GnT-V transfectants
Sugar chains at two glycosylation sites of IFN- 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)
using pyridylaminated (PA-) sugar chains (Table I). Reversed phase HPLC profiles of desialylated PA-sugar chains derived from IFN-
produced by CHO cells are shown in Figure 2. The proportion of each sugar chain is shown in Table III.
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Analysis of the branching degree of sugar chains
As described above, in IFN- 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-
was digested with ß-galactosidase (jack bean), endo-ß-galactosidase (Escherichia freundii) and
-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 GlcNAcß16 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 GlcNAcß16 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-
. From this analysis, it was revealed that more than half of the sugar chains linked to IFN-
were converted to the multiantennary types in the cells transfected with GnT-IV and/or GnT-V genes.
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The rate of sialylation
PA-sugar chains from IFN- produced by each CHO cell line were analyzed by anion exchange HPLC and the rate of sialic acid addition to IFN-
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-
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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Discussion |
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In contrast, we verified that the sugar chain structures of IFN- are changeable to multiantennary type, by increasing the GnT-IV and/or GnT-V expression level in IFN-
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-
: not because GnT-IV or GnT-V is hardly accessible to the sugar chains of IFN-
, 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- 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-
, 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-, 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-
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-
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- 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 ß13-N-acetylglucosaminyltransferase) for GlcNAcß16 branch (Van den Eijnden et al., 1988
). These results were consistent with previous reports (Yousefi et al., 1991
; Do and Cummings, 1993
).
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- 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-
of which sugar chains were remodeled to multiantennary structures.
It is reported that the sugar chains of IFN- influence the antiviral activity and pharmacokinetics in addition to physical properties such as protease resistance (Arakawa et al., 1986
; Bocci et al., 1985
; Sareneva et al., 1993
, 1994, 1995). It is therefore of interest to elucidate the properties imparted by each of the sugar chain structures of IFN-
. The work is in progress.
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Materials and methods |
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Culture of CHO cells
CHO cells producing human IFN- 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- 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- productivity
HIIF-D cell line producing human IFN- (obtained from ATCC, No. CRL-8200) was established from CHO cells deficient in dihydrofolate reductase (DHFR-) by cotransfection of pSV-IFN-
(containing the coding sequence for human IFN-
) and pAdD26SV(A)-3 (containing the coding sequence for the mouse DHFR) (Scahill et al., 1983
). HIIF-D subclone stably producing a high level of IFN-
, 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., 1998) 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., 1991
). 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., 1993
; Saito et al., 1994
). 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) with some modifications. Preparation of the substrate was done as reported (Tokugawa et al., 1996
). 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
-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-
IFN- was purified by immunoaffinity chromatography. The immunoaffinity column was prepared by coupling polyclonal anti-human IFN-
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 TrisHCl and 0.5 M NaCl, pH 7.5. After washing with CHROMATOP WASH BUFFER A (Nihon-gaishi, Japan), IFN-
was eluted with 0.2 M Gly-HCl, pH 2.5. The eluate was immediately neutralized with 1 M TrisHCl, pH 8.0 and concentrated and desalted by ultrafiltration with Ultrafree 15 centrifugal filter (Millipore).
Release of sugar chains from IFN- and pyridylamination
Purified IFN- 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 TrisHCl, 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). 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., 1988). 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)
. 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 -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)
. 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 -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
-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 420% SDSpolyacrylamide gradient gel by the method of Laemmli (Laemmli, 1970) 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 peroxidaseavidin (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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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