Reduction of the major xenoantigen on glycosphingolipids of swine endothelial cells by various glycosyltransferases

Masaru Koma2,3,4, Shuji Miyagawa1,3, Koichi Honke2, Yoshitaka Ikeda2, Souichi Koyota2, Shinichiro Miyoshi4, Hikaru Matsuda4, Shuichi Tsuji5, Ryota Shirakura3 and Naoyuki Taniguchi2

2Department of Biochemistry, 3Division of Organ Transplantation, Biomedical Research Center, 4Department of Surgery for Functional Regulation, Osaka University Graduate School of Medicine, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan, 5Molecular Glycobiology, Frontier Research Program, Inst. of Physical and Chemical Res. (RIKEN), Wako, Saitama 351–0100, Japan

Received on October 29, 1999; revised on January 17, 2000; accepted on January 19, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 The enzyme activities
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB4 (GSIB4) lectin, which binds to the Gal {alpha}1–3 Gal ß 1–4 GlcNAc-R ({alpha}-galactosyl epitope), was reduced by ~20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the {alpha}-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as {alpha}2,6-sialyltransferase (ST6Gal I), {alpha}2,3-sialyltransferase (ST3Gal III), and {alpha}1,2-fucosyltransferase ({alpha}1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the {alpha}-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and {alpha}1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and {alpha}1,2FT reduced the {alpha}-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.

Key words: glycosphingolipids/the {alpha}-galactosyl epitope/swine endothelial cell/xenotransplantation/glycosyltransferase


    Introduction
 Top
 Abstract
 Introduction
 Results
 The enzyme activities
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Genetic approaches to modify xenoantigens, such as Gal {alpha}1–3Gal ß1–4 GlcNAc-R ({alpha}-galactosyl epitope) (Galili et al., 1985Go, 1987, 1991), the major antigen in swine to human xenotransplantation, have been the goal of numerous xenotransplantation studies (Sandrin et al., 1995Go; Thall et al., 1995Go; Koike et al., 1996Go; Tearle et al., 1996Go; Tanemura et al., 1997Go, 1998).

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., 1993Go), GDP-L-fucose:ß-D-galactoside 2-{alpha}-L-fucosyltransferase ({alpha}1,2FT) (Larsen et al., 1990Go) and CMP-Sia:ß-D-Galß1–3(4)GlcNAc {alpha}2,3-sialyltransferase (ST3Gal III) (Kono et al., 1997Go), over UDP-Gal:ß-D-Galß1–4 GlcNAc {alpha}1,3- galactosyltransferase ({alpha}1,3GT) (Joziasse et al., 1989Go; Larsen et al., 1990Go), using transfected swine endothelial cell (SEC) lines.

In an earlier study, we classified the glycosyltransferases into two groups (Miyawaga et al., 1999Go). 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., 1996Go; Ihara et al., 1998Go). In addition, we have previously demonstrated the downregulation of xenoantigenicity by this enzyme, using SEC transfectants (Tanemura et al., 1997Go).

Group II includes glycosyltransferases which are known to participate in intracellular competition involving terminal glycosylation with {alpha}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-Galß1–4GlcNAc {alpha}2,6-sialyltransferase (ST6Gal I), and {alpha}1,2FT. ST6Gal I and ST3Gal III, for example, catalyze the glycosylation of N-acetyllactosamine residues in glycoproteins (Sandrin et al., 1995Go; Tanemura et al., 1998Go).

This study investigates the effect of group I and II glycosyltransferases on the {alpha}-galactosyl epitope, the glycosphingolipids of SEC in particular, using various transfected SEC lines.


    Results
 Top
 Abstract
 Introduction
 Results
 The enzyme activities
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
D-PDMP (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) treatment
The level of {alpha}-galactosyl epitope in glycosphingolipids contained by swine endothelial cells was investigated, using D-PDMP, which is a specific inhibitor of the glycosylation of glycosphingolipids (Inokuchi and Radin, 1987Go; Inokuchi et al., 1989Go).

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 {alpha}-galactosyl epitopes (Table I).


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Table I. FACS profiles of parental SEC and D-PDMP treated SEC which had been stained with NHS and GSIB4
 

    The enzyme activities
 Top
 Abstract
 Introduction
 Results
 The enzyme activities
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
An expression vector, which carried the cDNA of each glycosyltransferase was prepared. Transfection was performed using lipofectamine. Positive clones were established, and the enzyme activities therein were determined by high performance liquid chromatography (HPLC). Each established transfectant (Table II) shows the enzyme activity of the transfected gene.


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Table II. Enzyme activities of parental SEC and transfectants
 
Fluorescence histograms of parental SEC and transfectants
Flow cytometric profiles of parental SEC and transfectants which were stained with 20% normal human serum (NHS) and GSIB4 were prepared.

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 {alpha}-galactosyl epitopes were approximately 50–70% down-regulated in both groups of transfectants (Figure 1).



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Fig. 1. Flow cytometric analysis of transfectants stained with NHS or IB4 lectin. (A) Parental SEC (control) and stable transfectants were treated with 20% NHS as the first antibody and then FITC-conjugated anti-human Ig as a second antibody. (B) The reduction of the {alpha}–galactosyl epitope on cell surface was analyzed using GSIB4 lectin. Parental SEC (control) and stable transfectants were treated with FITC-conjugated GSIB4 lectin. Each value is expressed as the mean ± SD of four to six independent experiments.

 
Lactate dehydrogenase (LDH) assay of the transfected SEC
Amelioration of complement-mediated lysis by the transfection of glycosyltransferases was determined. In these experiments, NHS was used as a source of natural antibody to the {alpha}-galactosyl epitope and complement. The control parental SEC lysis resulting from 20% or 40% NHS treatment was found to be 23.1 ± 8.5% or 34.1 ± 7.5%, respectively (Figure 2A).




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Fig. 2. LDH assay of transfected SEC. (A) Amelioration of complement-mediated lysis by the transfectants and control parental SEC were estimated by 20% or 40% NHS which served as a source of natural antibody and complement. (B) The percent inhibition of complement-mediated lysis by transfectants. Each value is expressed as the mean ± SD of four to six independent experiments.

 
An ~50% inhibition in cytotoxicity was observed in the case of the group I transfectant, GnT-III, and 70–80% inhibition was in the group II transfectants, ST6Gal I, ST3Gal III, and {alpha}1,2FT. These results suggest that both types of glycosyltransferase are quite effective in reducing the {alpha}-galactosyl epitope level of SEC (Figure 2B).

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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Fig. 3. Western and lectin blotting. (A) Whole cell lysates from parental SEC and transfectants were separated by 12% SDS/PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with 0.2% of NHS. A typical Western blot pattern is shown. (B) A lectin blot analysis of whole cell lysates was also performed. The blots were probed with biotinylated GSIB4 lectin.

 
Thin layer chromatographic (TLC) analysis
Neutral glycosphingolipids were stained with the orcinol sulfuric acid reagent. A similar staining pattern was observed for parental SEC and all transfectants (Figure 4A). In a further step, the same glycosphingolipid fractions were separately developed and stained with NHS and GSIB4. Glycosphingolipids, which contained the {alpha}-galactosyl epitope were observed in a slow-migrating area under the Forssman antigen, indicating that they contain sugar chains larger than pentasaccharide. The reactivity for NHS and GSIB4 were reduced in the case of ST6Gal I, ST3Gal III, and {alpha}1,2FT transfectants but not the GnT-III transfectant (Figure 4B,C).




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Fig. 4. Orcinol staining of neutral glycosphingolipids and TLC immunostaining with NHS and GSIB4 lectin. (A) The total neutral glycosphingolipid fractions from parental SEC and transfectants were analyzed by thin layer chromatography. The marked area indicates glycosylceramide which contain more than five neutral sugar units in the glycosphingolipids. (B) Immunostaining of the neutral glycosphingolipids by NHS was performed in the lower area of TLC plate indicated by the mark. The quantitative analysis for the bands indicated by arrows a and b were done, using arbitrary units, in the bar graphs. Each value is expressed as the mean ± SD of three independent experiments. (C) Immunostaining of the {alpha}-galactosyl epitope in the neutral glycosphingolipids by GBIB4 lectin was performed in the lower area of TLC plate indicated by the mark. The quantitative analysis for the band indicated by arrows c was done, using arbitrary units, in the bar graphs. Each value is expressed as the mean ± SD of three independent experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 The enzyme activities
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The effect on the antigenicity in glycosphingolipids by various glycosyltransferases were assessed in this study. The reduction of antigenicity was initially tested by D-PDMP, which is a specific inhibitor against ceramide glucosyltransferase, and which was observed to abolish all the glycosylation of glycosphingolipids except for galactolipids. As expected, D-PDMP treatment reduced the reactivity of the parental SEC toward NHS and GSIB4 lectin by ~20%, suggesting that the glycosphingolipids in the SEC membrane contain meaningful amounts of xenoantigenicity to human, and especially {alpha}-galactosyl epitopes.

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., 1997Go, 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 50–80% suppression in the xenoantigen of SEC, although GnT-III acts only on N-linked sugars (Taniguchi et al., 1996Go) 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., 1997Go; Nakamura et al., 1997Go).

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 {alpha}2,6 sialyltransferase (Fast et al., 1993Go). 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., 1995Go). In addition, tunicamycin-treated GnT-III, which is not N-glycosylated, had almost no activity (Nagai et al., 1997Go). 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 {alpha}-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., 1996Go, 1997; Backer et al., 1998Go; Hallberg et al., 1998Go). Because of this, the immunostaining of this area in TLC was carried out. Staining with NHS and GSIB4 showed that the reduction in the {alpha}-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 {alpha}-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 {alpha}-galactosyl epitopes in glycosphingolipids, while group II glycosyltransferases, such as ST6Gal I, ST3Gal III, and {alpha}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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 The enzyme activities
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Endothelial cell culture
A swine endothelial cell (SEC) line, MYP30, was cultured in Dulbecco’s modified Eagle’s medium (D-MEM) containing 10% FBS with L-glutamine and penicillin/streptomycin (Gibco /BRL) (Miyagawa et al., 1994Go).

Construction of plasmids
cDNAs for mouse ST6Gal I and ST3Gal III were prepared. A cDNA of human {alpha}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 {alpha}1,2FT were subcloned into the site of pCXN2 (Niwa et al., 1991Go) 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., 1994Go). 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 {alpha}-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., 1984Go; Nishikawa et al., 1988Go; Kondo et al., 1990Go; Sasaki et al., 1993Go). 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 {alpha}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 {alpha}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 (GlcNAcß1–2Man{alpha}1–6 (GlcNAcß1–2Man{alpha}1–3) Manß1–4GlcNAcß1–4GlcNAc-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 manufacturer’s instructions, using a Kyokuto MTX "LDH" kit (Korzeniewski and Callewaert, 1983Go). 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, 1970Go) 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 peroxidase–conjugated 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 peroxidase–avidin 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., 1998Go; Ogiso et al., 1998Go).

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 {alpha}-galactosyl epitope. As a second treatment, the NHS treated TLC plate was treated with horseradish peroxidase–conjugated 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.).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 The enzyme activities
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. Milton S. Feather for his editing of the manuscript, and Mako Yamada for excellent technical assistance. This work was supported by Grants-in-Aid for Scientific Research on Priority Area No. 10178104 from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health and Welfare of Japan, and Program for Promotion of Basic Research Activities for Innovative Biosciences.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 The enzyme activities
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
the {alpha}–galactosyl epitope, the Gal {alpha}1–3 Gal ß1–4 GlcNAc-R; GnT-III, UDP-N-acetylglucosamine:ß-D-mannoside ß–1,4-N-acetylglucosaminyltransferase III; {alpha}1,2FT, GDP-L-fucose:ß-D-galactoside 2-{alpha}-L-fucosyltransferase; {alpha}1,3GT, UDP-Gal:ß-D-Galß1–4 GlcNAc {alpha}1,3- galactosyltransferase; ST3Gal III, CMP-Sia:ß-D-Galß1–3(4)GlcNAc {alpha}2,3- sialyltransferase; SEC, swine endothelial cell; ST6Gal I, CMP-Sia:ß-D-Galß1–4GlcNAc {alpha}2,6-sialyltransferase; D-PDMP, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; GSIB4, Griffonia simplicifolia I; HPLC, high performance liquid chromatography; NHS, normal human serum; LDH, lactate dehydrogenase; TLC, thin layer chromatography; FITC, fluorescein isothiocyanate.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 The enzyme activities
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Backer,A.E., Holgersson,J., Samuelsson,B.E. and Karlsson,H. (1998) Rapid and sensitive GC/MS characterization of glycolipid released Gal {alpha}1,3Gal-terminated oligosaccharides from small organ specimens of a single pig. Glycobiology, 8, 533–545.[Abstract/Free Full Text]

Bouhours,D., Pourcel,C. and Bouhours,J.E. (1996) Simultaneous expression by porcine aorta endothelial cells of glycosphingolipids bearing the major epitope for human xenoreactive antibodies (Gal {alpha}1–3Gal), blood group H determinant and N-glycolylneuraminic acid. Glycoconj. J., 13, 947–953.[ISI][Medline]

Bouhours,D., Liaigre,J., Naulet,J., Maume,D. and Bouhours,J.F. (1997) A novel glycosphingolipid expressed in pig kidney: Gal {alpha}1–3 Lewis (x) hexaglycosylceramide. Glycoconj. J., 14, 29–38.

Bouhours,J.F., Richard,C., Ruvoen,N., Barreau,N., Naulet,J. and Bouhours,D. (1998) Characterization of a polyclonal anti-Gal {alpha}1–3Gal antibody from chicken. Glycoconj. J., 15, 93–99.[ISI][Medline]

Fast,D.G., Jamieson,J.C. and McCaffrey,G. (1993) The role of the carbohydrate chains of Gal ß-1,4-GlcNAc {alpha} 2,6-sialyltransferase for enzyme activity. Biochim. Biophys. Acta., 1202, 325–330.[ISI][Medline]

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