2Department of Biochemistry, 3Division of Organ Transplantation, Biomedical Research Center, 4Department of Surgery for Functional Regulation, Osaka University Graduate School of Medicine, 22 Yamadaoka, Suita, Osaka 5650871, Japan, 5Molecular Glycobiology, Frontier Research Program, Inst. of Physical and Chemical Res. (RIKEN), Wako, Saitama 3510100, Japan
Received on October 29, 1999; revised on January 17, 2000; accepted on January 19, 2000.
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Abstract |
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Key words: glycosphingolipids/the -galactosyl epitope/swine endothelial cell/xenotransplantation/glycosyltransferase
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Introduction |
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Previous reports have noted the importance of the xenoantigen contained by the N-linked sugar and the dominance of the various glycosyltransferases, such as ß-D-mannoside ß1,4-N-acetylglucosaminyl transferase III (GnT-III) (Ihara et al., 1993), GDP-L-fucose:ß-D-galactoside 2-
-L-fucosyltransferase (
1,2FT) (Larsen et al., 1990
) and CMP-Sia:ß-D-Galß13(4)GlcNAc
2,3-sialyltransferase (ST3Gal III) (Kono et al., 1997
), over UDP-Gal:ß-D-Galß14 GlcNAc
1,3- galactosyltransferase (
1,3GT) (Joziasse et al., 1989
; Larsen et al., 1990
), using transfected swine endothelial cell (SEC) lines.
In an earlier study, we classified the glycosyltransferases into two groups (Miyawaga et al., 1999). Group I includes the glycosyltransferases which are responsible for remodeling the total glyco-antigen of the cell surface from core glycosylation and includes GnT-III. GnT-III catalyzes the branching of N-linked oligosaccharides to produce a bisecting N-acetylglucosamine (GlcNAc) residue. The attachment of a bisecting GlcNAc inhibits the further processing of oligosaccharides by other glycosyltransferases, such as N-acetylglucosaminyltransferase IV (GnT-IV) and N-acetylglucosaminyltransferase V (GnT-V) (Taniguchi et al., 1996
; Ihara et al., 1998
). In addition, we have previously demonstrated the downregulation of xenoantigenicity by this enzyme, using SEC transfectants (Tanemura et al., 1997
).
Group II includes glycosyltransferases which are known to participate in intracellular competition involving terminal glycosylation with 1,3GT for the common acceptor substrate in the trans Golgi stack and network and includes transferases such as ST3Gal III, CMP-Sia:ß-D-Galß14GlcNAc
2,6-sialyltransferase (ST6Gal I), and
1,2FT. ST6Gal I and ST3Gal III, for example, catalyze the glycosylation of N-acetyllactosamine residues in glycoproteins (Sandrin et al., 1995
; Tanemura et al., 1998
).
This study investigates the effect of group I and II glycosyltransferases on the -galactosyl epitope, the glycosphingolipids of SEC in particular, using various transfected SEC lines.
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Results |
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The use of D-PDMP led to a reduction in the reactivity of SEC to human natural antibodies and Griffonia simplicifolia IB4 (GSIB4) lectin by ~20%, suggesting that the glycosphingolipids in SEC contain a considerable amount of -galactosyl epitopes (Table I).
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The enzyme activities |
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While the control parental SEC and the mock cells reacted strongly with human natural antibodies in NHS and the GSIB4 lectin, the transfectants showed a diminished reactivity. The -galactosyl epitopes were approximately 5070% down-regulated in both groups of transfectants (Figure 1).
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Western and lectin blotting
Western and lectin blotting were performed, in order to analyze the alteration in reactivity of glycoproteins to human natural antibodies and the GSIB4 in the SEC transfectants. An evaluation of these blot profiles revealed that proteins derived from these transfectants with molecular masses below 66 kDa were responsible for the reduction in reactivity to NHS, compared to the parental SEC.
Similar to Western blotting patterns, proteins with molecular masses below 66 kDa in all the transfectants were responsible for the reduction in reactivity to GSIB4, as evidenced by lectin blotting (Figure 3).
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Discussion |
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In our previous study, we reported on an examination of the reduction of antigenicity of SEC via the overexpression of GnT-III or ST3Gal III (Tanemura et al., 1997, 1998). In this study, the overexpression of various glycosyltransferases including ST6Gal I were also tested, in terms of the down-regulation of the expression of the xenoantigen on SEC. The issue that these approaches are not completely effective, however, remains. These glycosyltransferases achieved a 5080% suppression in the xenoantigen of SEC, although GnT-III acts only on N-linked sugars (Taniguchi et al., 1996
) and ST3Gal III acts mainly, but not exclusively, on N-linked sugars. One explanation of this result is that these glycosyltransferases act, not only on N-linked sugars, but also on O-linked sugars and glycosphingolipids either directly or indirectly.
A direct effect on glycosphingolipids by GnT-III is unlikely, whereas those by the overexpressed ST6Gal I and ST3Gal III are conceivable (Kono et al., 1997; Nakamura et al., 1997
).
On the other hand, the indirect effect on glycosphingolipids by each glycosyltransferase was, to some extent, expected. It has been reported that the presence of the trimannose core with GlcNAc attached is important for the expression of the catalytic activity of 2,6 sialyltransferase (Fast et al., 1993
). Other investigations have also reported that N-linked carbohydrates on ß1,4N-acetylgalactosaminyltransferase are required for regulating the stability of the enzyme structure (Haraguchi et al., 1995
). In addition, tunicamycin-treated GnT-III, which is not N-glycosylated, had almost no activity (Nagai et al., 1997
). These reports raise the possible conclusion that each of these glycosyltransferases are closely related. Therefore, glycosyltransferases such as ST6Gal I and ST3Gal III might affect glycosphingolipids via the modulation of glycosyltransferases which act on glycophingolipids directly.
The -galactosyl epitopes in glycosphingolipids of swine cells have already been reported in several papers. They were contained in excess of the pentaglycosylceramide of neutral glycosphingolipids (Bouhours et al., 1996
, 1997; Backer et al., 1998
; Hallberg et al., 1998
). Because of this, the immunostaining of this area in TLC was carried out. Staining with NHS and GSIB4 showed that the reduction in the
-galactosyl epitopes of neutral glycosphingolipids was not observed in GnT-III but, rather, in group II transferase-transfected SECs. The overexpressed ST6Gal I and ST3Gal III downregulated the antigenicity of glycosphingolipid of SEC.
Regarding ST6Gal I, this glycosyltransferase might be more effective in reducing the xenoepitope of both glycoprotein and glycosphingolipids of SEC than ST3Gal III. However, differences between these glycosyltransferases, as evidenced by Western blot and TLC immunostaining, were not detected in this study.
In conclusion, we showed that both groups of glycosyltransferases significantly downregulated the -galactosyl epitopes of the total cell surface as judged by flow cytometric analyses and a cytotoxicity assay. GnT-III caused only small alterations in the
-galactosyl epitopes in glycosphingolipids, while group II glycosyltransferases, such as ST6Gal I, ST3Gal III, and
1,2FT, showed a significant level of reduction. Further studies are under way to determine the differences among these transferases, via the use of transgenic mice.
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Materials and methods |
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Construction of plasmids
cDNAs for mouse ST6Gal I and ST3Gal III were prepared. A cDNA of human 1,2FT was a gift from Dr. John B.Lowe (University of Michigan). The cDNAs for human GnT-III, mouse ST6Gal I and ST3Gal III, and human
1,2FT were subcloned into the site of pCXN2 (Niwa et al., 1991
) in which the transcription of the inserted cDNA is driven by a ß-actin promoter and a cytomegalovirus enhancer. A neomycin-resistant gene contained in the vector permitted the selection of the transfectants by the antibiotic, G418. The resulting plasmids were purified by CsCl-gradient ultracentrifugation, and used directly for transfection.
Transfection experiments
The purified plasmids (20 µg) were introduced into MYP-30 by lipid-mediated DNA transfection using a lipofectamine reagent (GIBCO/BRL). The transfected MYP-30 cells were maintained in a complete medium for several days in an atmosphere of humidified 5% CO2 at 37°C and were then transferred to a complete medium containing 0.4 mg/ml G418 (GIBCO/BRL) for selection (Miyagawa et al., 1994). Expression of the glycosyltransferases were confirmed by activity assays, as described below.
D-PDMP treatment
Parental SECs were treated with 20 µM D-PDMP for 40 h in D-MEM containing 10% FBS. The D-PDMP treated SEC were then incubated with 20% NHS and GSIB4 lectin (EY Laboratories Co. Ltd., USA) which binds to the -galactosyl epitope. The reduced reactivity of SEC to NHS and GSIB4 by D-PDMP treatment was analyzed by flow cytometry.
Glycosyltransferase assays
The glycosyltransferase activities in each transfectant were assayed using fluorescence-labeled oligosaccharides as the acceptor substrates by a reversed-phase HPLC system equipped with a fluorescence detector (Hase et al., 1984; Nishikawa et al., 1988
; Kondo et al., 1990
; Sasaki et al., 1993
). For the enzyme activity assays, cells were washed twice with PBS and then centrifuged at 1500 x g for 10 min. The pelleted cells were resuspended in 100 µl of PBS and then lysed by sonication. The resulting lysates were then assayed for activity. In the activity assays for ST6Gal I, ST3Gal III, and
1,2FT, pyridylaminated lacto-N-neotetraose (LNnT-PA, Seikagaku Kogyo), a common acceptor substrate, was used. To determine these enzyme activities, whole cell lysates were incubated at 37°C for 3 h in the reaction mixtures. The mixtures used were 50 mM cacodylate buffer, 10 mM MnCl2, 0.23% Triton X-100, 5 mM CMP-sialic acid as the donor substrate and 10 mM LNnT-PA as the acceptor, pH 6.8, for ST6Gal I and ST3Gal III; 13 mM potassium phosphate buffer, 0.1% Triton X-100, 13 mM phenyl-ß-D-galactoside, 5 mM ATP, 1 mM GDP-fucose, and 10 mM LNnT-PA, pH 6.1, for
1,2FT. When the activity of GnT-III was assayed, the cell lysates were reacted with 20 mM UDP-GlcNAc and 20 mM the pyridylaminated agalacto biantennary oligosaccharide (GlcNAcß12Man
16 (GlcNAcß12Man
13) Manß14GlcNAcß14GlcNAc-PA) in the presence of 63 mM MES, 10 mM MnCl2, 200 mM GlcNAc, and 0.5% Triton X-100, pH 6.25. The assay mixtures were incubated at 37°C for 3 h, and the reactions were then terminated by heating in a boiling water bath for 5 min, followed by centrifugation of the samples at 15,000 x g for 5 min. The resulting supernatants were injected into a reversed phase HPLC equipped with a TSKgel column, ODS 80TM (4.6 x 250 mm). The product and the substrate were isocratically separated with 20 mM ammonium acetate buffer (pH 4.0) containing 0.01% n-butanol. The fluorescence of the column elute was detected with a fluorescence detector (Shimazu, model RF-10AXL) using excitation and emission wavelengths of 320 nm and 400 nm, respectively. The specific activity of the enzyme is expressed as picomoles of product produced per hour of incubation per milligram of protein. Protein concentrations were determined with a BCA protein assay kit (Pierce), using bovine serum albumin as a standard.
Flow cytometry
Parental SEC, D-PDMP treated SEC, and transfectants were incubated with 20% NHS at 4°C for 1 h, washed, and then incubated with 1.25 mg of fluorescein isothiocyanate (FITC)-conjugated anti-human Ig (Cappel) as a second antibody for 1 h at 4°C. Stained cells were analyzed with a FACS Calibure flow cytometer (Becton Dickinson). The direct fluorescence of cell-surface carbohydrate epitopes was also examined with a FITC-conjugated GSIB4 lectin.
LDH assay
This assay was performed according to the manufacturers instructions, using a Kyokuto MTX "LDH" kit (Korzeniewski and Callewaert, 1983). The transfected cells were plated at 2 x 104 cells per well in 96-well trays 1 day prior to assay. The next morning, the wells were washed twice in serum-free D-MEM to remove the LDH which is present in fetal calf serum, and incubated in several concentrations of NHS which had been diluted with D-MEM. The plates were incubated for 2 h at 37°C and the released LDH was then measured. The percent cytotoxicity was calculated using the formula:
Cytotoxicity = E N S / M N S x 100,
where E is the experimentally observed release of LDH activity from the target SEC, N the LDH activity in each concentration of NHS, S the spontaneous release of LDH activity from target SEC incubated in the absence of NHS, and M the maximal release of LDH activity, as determined by sonication.
Western blotting
Total cell lysates (10 µg) from parental or transfected SEC were subjected to 12% SDS/PAGE under reducing conditions using the methods of Laemmli (Laemmli, 1970) and then transferred electrophoretically onto a nitrocellulose membrane (Schleicher & Schuell). The membrane was blocked in PBS containing 3 % BSA and incubated for 20 min with 0.2% NHS at room temperature. After washing, the blots were incubated with horseradish peroxidaseconjugated rabbit Fab to human immunoglobulins (IgG, IgM, IgA; Cappel) and developed using an ECL detection system (Amersham Pharmacia Biotech).
Lectin blotting
Parental and transfected cell products were also tested by lectin blot analysis, using GSIB4. The cell lysate (10 µg) was subjected to 12% SDS-PAGE under reducing conditions and then transferred electrophoretically onto a nitrocellulose membrane (Schleicher & Schuell). The blots were blocked in PBS containing 0.05% Tween 20 and 3% BSA and incubated for 20 min with biotinylated 10 µg /ml GSIB4. After washing, the blots were incubated with horseradish peroxidaseavidin complex (Vector, ABC Reagent) and developed using an ECL detection system (Amersham Pharmacia Biotech).
Preparation of glycosphingolipids
Total glycosphingolipid fractions were isolated from SEC (1 x 108 cells) using the Folch method. The following steps involved the use of DEAE-Sephadex A-25-columns (Pharmacia LKB Biotech) to separate the acidic glycosphingolipids from the neutral glycosphingolipid components. Neutral glycosphingolipids were partially purified by mild alkaline treatment. Acidic glycosphingolipid were applied to a SEP-PAKC18 cartridge (Waters Associates, Milford, MA) to remove salts.
Thin layer chromatography (TLC)
Glycosphingolipids were separated on precoated silica gel Merck 60 plates (HPTLC plates, purchased from E.Merck, Darmstadt, Germany). Solvent systems were as follows: A, chloroform/methanol/water (60/35/8, v/v/v) for neutral glycosphingolipids; B, chloroform/methanol/0.2% CaCl2 (60/40/9, v/v/v) for acidic glycosphingolipids. Neutral and acidic glycosphingolipids were visualized by spraying the TLC-plate with orcinol/sulfuric acid reagent (Bouhours et al., 1998; Ogiso et al., 1998
).
TLC immunostaining
The total neutral glycosphingolipid fractions from parental SEC and transfectants were separated by thin layer chromatography followed by immunostaining with human natural antibody or biotinylated GSIB4 lectin in order to identify the -galactosyl epitope. As a second treatment, the NHS treated TLC plate was treated with horseradish peroxidaseconjugated rabbit Fab to human immunoglobulins (IgG, IgM, IgA) (Cappel). Lectin blot analysis was also performed using the Vector ABC reagent (Vector Laboratories). The ECL detection system (Amersham Pharmacia Biotech) was employed for the development of both stainings. Bands on the immunostaining were evaluated by scanning with a Scanning Imager (Molecular Dynamic Co., Ltd.).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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