Absorption of anti–blood group A antibodies on P-selectin glycoprotein ligand-1/immunoglobulin chimeras carrying blood group A determinants: core saccharide chain specificity of the Se and H gene encoded {alpha}1,2 fucosyltransferases in different host cells

Jonas C. Löfling1, Elenor Hauzenberger and Jan Holgersson

Division of Clinical Immunology, F79, IMP1, Karolinska Institutet, Huddinge University Hospital AB, S-141 86 Stockholm, Sweden

Received on August 6, 2001; revised on October 25, 2001; accepted on November 2, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
To specifically eliminate recipient anti-blood group ABO antibodies prior to ABO-incompatible organ or bone marrow transplantation, an efficient absorber of ABO antibodies has been developed in which blood group determinants may be carried at high density and by different core saccharide chains on a mucin-type protein backbone. The absorber was made by transfecting different host cells with cDNAs encoding a P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b chimera (PSGL-1/mIgG2b), the H- or Se-gene encoded {alpha}1,2-fucosyltransferases (FUT1 or FUT2) and the blood group A gene encoded {alpha}1,3 N-acetylgalactosaminyltransferase ({alpha}1,3 GalNAcT). Western blot analysis of affinity-purified recombinant PSGL-1/mIgG2b revealed that different precursor chains were produced in 293T, COS-7m6, and Chinese hamster ovary (CHO)-K1 host cells coexpressing FUT1 or FUT2. FUT1 directed expression of H type 2 structures mainly, whereas FUT2 preferentially made H type 3 structures. None of the host cells expressing either FUT1 or FUT2 supported expression of H type 1 structures. Furthermore, the highest A epitope density was on PSGL-1/mIgG22b made in CHO-K1 cells coexpressing FUT2 and the {alpha}1,3 GalNAcT. This PSGL-1/mIgG2b was used for absorption of anti–blood group A antibodies in human blood group O serum. At least 80 times less A trisaccharides on PSGL-1/mIgG2b in comparison to A trisaccharides covalently linked to macroporous glass beads were needed for the same level of antibody absorption. In conclusion, PSGL-1/mIgG2b, if substituted with A epitopes, was shown to be an efficient absorber of anti–blood group A antibodies and a suitable model protein for studies on protein glycosylation.

Key words: ABO incompatible/alpha1,2-fucosyltransferase/antibody absorption/glycoconjugate/mucin


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The major human blood group system, the histo-blood group ABO system, is defined by three carbohydrate determinants, the blood group A, B, and H epitopes. The H epitope, Fuc{alpha}2GalßR, is the immediate precursor for the A and B epitope–forming enzymes. An {alpha}1,2 fucosyltransferase (FT) catalyzes the reaction between Galß-R and GDP-Fuc. Hitherto, two loci have been identified in humans coding for enzymes catalyzing this reaction, the products of the H (FUT1) and Se (FUT2) genes respectively (Larsen et al., 1990Go; Kelly et al., 1995Go; Rouquier et al., 1995Go). The blood group A gene encodes an {alpha}1,3 N-acetylgalactosaminyltransferase ({alpha}1,3 GalNAcT) and the B gene an {alpha}1,3 galactosaminyltransferase ({alpha}1,3 GalT) (Yamamoto et al., 1990Go), which transfer an {alpha}-GalNAc and an {alpha}-Gal residue, respectively, to the third carbon of the terminal galactose of the H determinant.

Glycans carrying the ABH determinants are found on glycoproteins, on glycolipids, or as free oligosaccharides. ABH antigens can be found on N- or O-linked glycans. The most common core structures described so far on O-linked glycans are core 1 (Galß3GalNAc), core 2 (Galß3(GlcNAcß6)GalNAc), core 3 (GlcNAcß3GalNAc), and core 4 (GlcNAcß3(GlcNAcß6)GalNAc). These have been shown to carry type 1 (Galß3GlcNAc), type 2 (Galß4GlcNAc), and type 3 (Galß3GalNAc{alpha}) structures (Takasaki et al., 1978Go; Donald, 1981Go; Clausen and Hakomori, 1989Go; Holgersson et al., 1992Go; Hakomori, 1999Go; Lowe, 1999Go). Type 1 structures are mainly found as extensions of the core 3 and 4 structures, whereas type 2 chains (polylactosamine) are seen as extensions on the GlcNAcß1,6 branch of core 2 structures (Clausen and Hakomori, 1989Go; Lowe, 1999Go).

There are numerous reports on the results of either transfection of different cells with the different {alpha}1,2 FTs (Ernst et al., 1989Go; Rajan et al., 1989Go; Prieto et al., 1995Go; Liu et al., 1998Go, 1999; Zerfaoui et al., 2000Go), or on the specificity in vitro of the {alpha}1,2 FTs with synthetic substrates (Beyer et al., 1980Go; Kumazaki and Yoshida, 1984Go; Betteridge and Watkins, 1985Go; Ernst et al., 1989Go; Rajan et al., 1989Go; Kyprianou et al., 1990Go; Larsen et al., 1990Go; Sarnesto et al., 1990Go, 1992). However, less is known about the synthesis of the various ABH antigens by these enzymes in different cell types on a defined carrier protein. There is one study that was undertaken investigating the substrate specificity of the H type {alpha}1,2 FT on various glycoprotein acceptors (Prieto et al., 1997Go). As far as we know, there has not been any equivalent study on the Se gene encoded {alpha}1,2 FT or a comparison of the action of the two {alpha}1,2 FTs on one single glycoprotein produced in different cell lines.

Transplantation (Tx) across the ABO barrier is usually avoided in organ Tx because the risk of antibody-mediated rejection (AMR) due to preformed antibodies is often high (Porter, 1963Go; Gugenheim et al., 1990Go; Sanchez-Urdazpal et al., 1993Go; Farges et al., 1995Go). This may also hold true in bone marrow transplantation (Benjamin and Antin, 1999Go; Benjamin et al., 1999Go), though it has long been the belief that blood group ABH incompatibility does not affect the outcome (Gale et al., 1977Go). However, in some cases it would still be desirable to transplant across the ABO barrier, one of the reasons being that it widens the pool of available donors for a particular recipient, even if it does not increase the total number of donors (Gugenheim et al., 1990Go; Farges et al., 1995Go; Gibbons et al., 2000Go).

Removal of anti-A or anti-B antibodies by extracorporeal immunoabsorption (EIA) or plasmapheresis (PP) has been shown to improve graft survival following ABO-incompatible organ Tx (Eid et al., 1998Go; Tamaki et al., 1998Go; Alkhunaizi et al., 1999Go). Another method used for the prevention of AMR in both ABO-incompatible Tx and xeno-Tx is infusion of free oligosaccharides (Cooper et al., 1993Go; Simon et al., 1998Go), but low affinity of antibodies for free saccharides (Galili and Matta, 1996Go) and the short half-life of low-molecular-weight oligosaccharides in the circulation (Ye et al., 1994Go; Simon et al., 1998Go) may prevent a wider use. Synthetic immunoabsorbents for extracorporeal absorption of anti–blood group antibodies are available (Rieben et al., 1995Go) and have been used both in animal models and clinically (Bensinger, 1981Go; Bensinger et al., 1981aGo,b, 1982).

Here, we describe the production in various host cells of a recombinant P-selectin glycoprotein-1/mouse IgG2b (PSGL-1/mIgG2b) chimera substituted with blood group H and A determinants. The repertoire of core saccharide structures carrying H epitopes was investigated by western blotting using core-specific anti-H monoclonal antibodies. The A epitope density was correlated to the type of host cell used for transfection and to the {alpha}1,2 FT gene used to obtain H precursor chains. Furthermore, the ability of blood group A substituted PSGL-1/mIgG2b chimeras to absorb anti–blood group A antibodies from human blood group O serum was evaluated.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Blood group H and A determinants on recombinant PSGL-1/mIgG2b made in various host cells
293T cells.
Immunoaffinity-purified PSGL-1/mIgG2b produced by 293T cells transiently transfected with the PSGL-1/mIgG2b cDNA with and without the plasmids encoding the H, Se, or A genes was analyzed by sodium dodecyl sulfate–polyacrylamide gel elecrophoresis (SDS–PAGE) and western blot using anti-mIgG, anti-blood group A, H type 1, H type 2, and H type 3 specific antibodies for detection (Figure 1A–E). The fusion protein migrated under reducing conditions as a doublet with an apparent molecular weight of approximately 100 and 140 to 160 kDa (A). The PSGL-1/mIgG2b stained poorly with silver (not shown) in concordance with previous observations with respect to the behavior of highly glycosylated, mucin-type proteins (Carraway and Hull, 1991Go; Shimizu and Shaw, 1993Go). As shown before (Liu et al., 1997Go), the fusion protein was produced as a homodimer (data not shown). Double bands around 65 kDa and 35 kDa were detected with the anti-mIgG antibody. These bands were not seen in the supernatants of 293T cells transfected with empty vector alone and are thus likely to be proteolytic fragments derived from the fusion protein (Carraway and Hull, 1991Go). The 47-kDa and 20-kDa bands seen in cell fractions are derived from the heavy and light Ig chains of the goat anti-mouse antibody, which is coming off the agarose beads on boiling in sample buffer.



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Fig. 1. SDS–PAGE and western blot analysis of immunopurified PSGL-1/mIgG2b chimeras produced in 293T cells transfected with the H or Se gene alone, or in combination with the A gene encoded {alpha}1,3 GalNAcT. Following separation on an 8% SDS–PAGE and blotting onto nitrocellulose membranes, the PSGL-1/mIgG2b chimeras were probed with an anti-mouse IgG antibody (A), an anti–blood group A antibody followed by a goat anti-mouse IgM antibody (B), an anti-H type 1 chain-specific antibody followed by a HRP-labelled goat anti-mouse IgG3 antibody (C), an anti-H type 2 chain–specific antibody followed by a goat anti-mouse IgM-HRP antibody (D), and an anti-H type 3 chain–specific antibody followed by a HRP-labeled goat anti-mouse IgM antibody (E). In panels AD, samples analyzed were from cells transfected with plasmids encoding CDM8 (lanes 1 and 5), PSGL-1/mIgG2b (lanes 2 and 6), PSGL-1/mIgG2b and the H gene (lane 3), PSGL-1/mIgG2b, the H and A gene (lane 4), PSGL-1/mIgG2b and the Se gene (lane 7), or PSGL-1/mIgG2b and the Se and A gene (lane 8). In E the duplicate samples from CDM8 and PSGL-1/mIgG2b transfected cells were omitted. In C, 250 ng of H-type 1 chain-BSA was used as a positive control.

 

Both FUT1 and FUT2 could with the {alpha}1,3 GalNAcT support the biosynthesis of blood group A chains on the PSGL-1/mIgG2b (B). The combination of FUT2 and the {alpha}1,3 GalNAcT seemed to create more A epitopes on PSGL-1/mIgG2b than the combination of FUT1 and the {alpha}1,3 GalNAcT (cf. these lanes in B). On the other hand, FUT1 alone supported expression of abundant H type 2 structures (D), whereas FUT2 gave rise to few H type 2 epitopes on the mucin/Ig (D). There were no detectable H type 1 structures on PSGL-1/mIgG2b (C). The H epitopes created by FUT2 were based almost exclusively on type 3, that is, Fuc{alpha}2Galß3GalNAc{alpha}-R, because no H type 1 structures and few H type 2 structures were made on PSGL-1/mIgG2b by this enzyme (E). Interestingly, cotransfection of both the H or Se gene with the A gene gave rise to abundant epitopes on the PSGL-1/mIgG2b reactive with the anti-H type 3 antibody (E). Blood group A, H type 2 and 3 epitopes were mainly O-linked, because peptide:N-glycosidase F (PNGaseF) treatment of PSGL-1/mIgG2b did not decrease antibody staining using antibodies specific for these epitopes (Figure 2B–D). Efficient N-glycan deglycosylation was indicated by a complete mobility shift of the PSGL-1/mIgG2b (Figure 2A–D).



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Fig. 2. SDS–PAGE and western blot analysis of PNGaseF-treated immunopurified PSGL-1/mIgG2b produced in 293T cells transfected with the H or Se gene alone or in combination with the A gene encoded {alpha}1,3 GalNAcT. Following PNGaseF treatment (+), or not (–), of immunopurified PSGL-1/mIgG2b, the PSGL-1/mIgG2b was separated on an 8% SDS–PAGE and blotted onto nitrocellulose membranes. PSGL-1/mIgG2b chimeras were probed with an anti-mouse IgG antibody (A), an anti–blood group A antibody followed by a goat anti-mouse IgM antibody (B), an anti-H type 2 chain–specific antibody followed by a goat anti-mouse IgM-HRP antibody (C), and an anti-H type 3 chain–specific antibody followed by an HRP-labeled goat anti-mouse IgM antibody (D). In panels AD, samples analyzed were from cells transfected with plasmids encoding PSGL-1/mIgG2b and the H gene (lanes 1 and 2), PSGL-1/mIgG2b and the H and A gene (lanes 3 and 4), PSGL-1/mIgG2b and the Se gene (lanes 5 and 6), or PSGL-1/mIgG2b and the Se and A gene (lanes 7 and 8).

 
COS cells.
The western blot analysis of PSGL-1/mIgG2b made in COS cells is shown in Figure 3A–E. Although weaker, the pattern of PSGL-1/mIgG2b staining using anti-A and H specific antibodies was somewhat similar to that found for the PSGL-1/mIgG2b made in 293T cells. Both FUT1 and FUT2 could together with the {alpha}1,3 GalNAcT support A epitope expression, with more epitopes created by FUT2 and the {alpha}1,3 GalNAcT (B). Only FUT1 could make H type 2 structures (D), and neither FUT1 nor FUT2 supported expression of H type 1 structures in COS cells (C). In contrast to the PSGL-1/mIgG2b made in 293T-cells, very low levels of H type 3 epitopes were seen on the PSGL-1/mIgG2b produced in COS cells coexpressing FUT1 and the {alpha}1,3 GalNAcT, or the FUT2 enzyme alone (E). However, joint expression of FUT2 and the {alpha}1,3 GalNAcT gave rise to increased reactivity with the H type 3 antibody (E). Furthermore, weak bands were seen with the anti-A antibody when only the {alpha}1,2 FTs were expressed, indicating a weak endogenous activity of an {alpha}1,3 GalNAcT in COS cells, as previously reported (Clarke and Watkins, 1999Go). PNGaseF treatment of PSGL-1/mIgG2b did not show any detectable reduction in anti–blood group A, H type 2 or 3 antibody staining (data not shown), indicating that these epitopes are mainly carried on PSGL-1/mIgG2b O-glycans.



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Fig. 3. SDS–PAGE and western blot analysis of immunopurified PSGL-1/mIgG2b chimeras produced in COS-7m6 cells transfected with the H or Se gene alone or in combination with the A gene encoded {alpha}1,3 GalNAcT. Following separation on an 8% SDS–PAGE and blotting onto nitrocellulose membranes, the PSGL-1/mIgG2b chimeras were probed with an anti-mouse IgG antibody (A), an anti–blood group A antibody followed by a goat anti-mouse IgM antibody (B), an anti-H type 1 chain–specific antibody followed by an HRP-labeled goat anti-mouse IgG3 antibody (C), an anti-H type 2 chain–specific antibody followed by a goat anti-mouse IgM-HRP antibody (D), and an anti-H type 3 chain–specific antibody followed by a HRP-labeled goat anti-mouse IgM antibody (E). In panels AD, samples analyzed were from cells transfected with plasmids encoding CDM8 (lanes 1 and 5), PSGL-1/mIgG2b (lanes 2 and 6), PSGL-1/mIgG2b and the H gene (lane 3), PSGL-1/mIgG2b and the H and A gene (lane 4), PSGL-1/mIgG2b and the Se gene (lane 7), or PSGL-1/mIgG2b and the Se and A gene (lane 8). In E the duplicate samples from CDM8 and PSGL-1/mIgG2b transfected cells were omitted. In C, 250 ng of H-type 1 chain-BSA was used as a positive control.

 

Chinese hamster ovary (CHO) cells.
Figure 4A–E, shows the staining of PSGL-1/mIgG2b made in CHO cells. PSGL-1/mIgG2b carried blood group A epitopes following coexpression of FUT1 or FUT2 cDNAs with the {alpha}1,3 GalNAcT (B). Neither H type 1 (C) nor H type 2 (D) structures could be detected on the PSGL-1/mIgG2b chimera made in CHO cells following cotransfection with either FUT1 or FUT2, suggesting that H type 3 structures are the sole precursors available for the {alpha}1,3 GalNAcT. Staining with the anti-H-type 3 antibody was seen with both FUT1 and FUT2 (E), supporting the theory that the A epitopes are based solely on core 1 structures in CHO cells. Further support to this was obtained after PNGaseF treatment, which did not affect the antibody staining intensity, indicating an O-glycan restricted A epitope expression (data not shown). The number of A epitopes and H type 3 epitopes on the PSGL-1/mIgG2b chimera were clearly higher with FUT2 as compared to FUT1 suggesting that the Se gene product is superior to the H gene product in terms of {alpha}1,2-fucosylation of core 1 (Galß3GalNAc{alpha}-Ser/Thr) structures.



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Fig. 4. SDS–PAGE and western blot analysis of immunopurified PSGL-1/mIgG2b chimeras produced in CHO-K1 cells transfected with the H or Se gene alone or in combination with the A_gene encoded {alpha}1,3 GalNAcT. Following separation on an 8% SDS–PAGE and blotting onto nitrocellulose membranes, the PSGL-1/mIgG2b chimeras were probed with an anti-mouse IgG antibody (A), an anti–blood group A antibody followed by a goat anti-mouse IgM antibody (B), an anti-H type 1 chain–specific antibody followed by an HRP-labeled goat anti-mouse IgG3 antibody (C), an anti-H type 2 chain–specific antibody followed by a goat anti-mouse IgM-HRP antibody (D), and an anti-H type 3 chain–specific antibody followed by an HRP-labeled goat anti-mouse IgM antibody (E). Samples analyzed were from cells transfected with plasmids encoding CDM8 (lane 1), PSGL-1/mIgG2b (lane 2), PSGL-1/mIgG2b and the H gene (lane 3), PSGL-1/mIgG2b and the H and A gene (lane 4), PSGL-1/mIgG2b and the Se gene (lane 5), or PSGL-1/mIgG2b and the Se and A gene (lane 6). In C, 250 ng of H-type 1 chain-BSA was used as a positive control.

 

The relative blood group A epitope density on PSGL-1/mIgG2b produced in different host cells
To semi-quantify the relative number of A epitopes on the PSGL-1/mIgG2b chimera made in various host cells expressing the A gene with either of the {alpha}1,2 FTs, western blotting with anti-A antibodies and anti-mIgG antibodies followed by chemiluminescence detection in a Fluor-S®Max MultImager was used. The ratios of the blood group A and the mIgG reactivities for each PSGL-1/mIgG2b are shown in Figure 5 (one of three representative experiments shown). As seen, the A epitope density was highest on the PSGL-1/mIgG2b made in CHO cells expressing the A gene together with the Se gene encoded {alpha}1,2 FT. This PSGL-1/mIgG2b carried approximately three times more A epitopes than the PSGL-1/mIgG2b made in 293T cells transfected with FUT2 and the A gene; the PSGL-1/mIgG2b having the second highest A epitope density (Figure 5). In each cell line, the FUT2 gave higher A epitope density together with the A gene than did FUT1 together with the A gene.



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Fig. 5. The relative blood group A density on recombinant PSGL-1/mIgG2b produced in COS, CHO, and 293T cells coexpressing FUT1 or FUT2 with {alpha}1,3 GalNAcT. The density was calculated as the ratio of the volumes for the blood group A and the mIgG reactivities as described in Materials and methods, and the values were normalized to the ones obtained for the A substituted PSGL-1/mIgG2b produced in COS cells coexpressing FUT1 and the {alpha}1,3 GalNAcT.

 
Absorption of anti-A antibodies on PSGL-1/mIgG2b carrying blood group A epitopes
The efficacy of anti-A antibody absorption on recombinant PSGL-1/mIgG2b with or without blood group A epitopes was compared to that of absorption on A trisaccharides linked via poly[N-(2-hydroxyethyl)acrylamide] to macroporous glass beads (A-PAA-MPG). Pre- and postabsorption anti-A-antibody levels were assessed in an enzyme-linked immunosorbent assay (ELISA) in which the plate was coated with A trisaccharides linked to poly[N-(2-hydroxyethyl)acrylamide also substituted with biotin (A-PAA-biotin). The results are shown in Fig 6. Twenty pmol of recombinant A epitope-substituted PSGL-1/mIgG2b were needed to absorb 60% of the anti-A antibodies as detected in the A-PAA-biotin ELISA, whereas 164,000 pmoles of A determinants as A-PAA-MPG were needed to absorb the same amount of anti-A antibodies. The PSGL-1/mIgG2b dimer has 106 potential O-linked glycosylation sites and eight potential N-linked glycosylation sites (Wilkins et al., 1996Go; Aeed et al., 1998Go, 2001), of which the latter eight may carry branched structures. If one assumes that each PSGL-1/mIgG2b carries approximately 100 A epitopes, which most likely is an overestimation, the mucin made in CHO cells with the FUT2 and the {alpha}1,3 GalNAcT is approximately 80 times more efficient on a carbohydrate molar basis than is A-PAA-MPG.



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Fig. 6. Anti–blood group A reactivity remaining in serum after absorption on different blood group A substituted absorbents. Anti–blood group A antibodies were absorbed as described in Materials and methods. The remaining reactivity, in percentage of nonabsorbed blood group O serum, as measured by an ELISA coated with A-PAA-biotin, was plotted against the amount of absorber used. A trend line was calculated using logarithmic regression. The circles represent PSGL-1/mIgG2b produced in CHO cells with FUT2 and {alpha}1,3 GalNAcT, and the squares represent A trisaccharides linked via A-PAA-MPG. B trisaccharides coupled via B-PAA-MPG were used to obtain the same amount of PAA-MPG in all absorptions.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Studies in vitro of FUT1 and FUT2 have shown that the enzymes are able to use a wide range of substrates, including core 1 and lactosamine structures. In CHO cells stably expressing FUT1, {alpha}1,2-fucosylation on glycoproteins was only directed to those containing polylactosamine sequences (Prieto et al., 1997Go). Thus {alpha}1,2-fucosylation on glycoproteins by FUT1 was considered to occur solely on N-linked glycans in CHO cells because there are no core 2 structures, which are necessary for polylactosamine structure formation on O-linked glycans (Prieto et al., 1997Go). Furthermore, experiments in CHO cells have indicated that only a relatively small proportion of N-linked glycans on glycoproteins was modified by {alpha}1,2-fucosylation (Prieto et al., 1997Go; Zerfaoui et al., 2000Go).

For elucidation of the mechanisms behind precursor protein selectivity, a model glycoprotein (such as our recombinant PSGL-1/mIgG2b) can be very useful. Our experiments show that the glycosylation of recombinant PSGL-1/mIgG2b does indeed correlate with in vitro studies. FUT1 has a marked preference for type 2 precursor chains, whereas FUT2 clearly prefers type 3/core 1 structures. However, as seen in Figure 1D, FUT2 is able (though rather poorly) to use type 2 chains as precursors for fucosylation of glycoproteins, as has been suggested by studies on the substrate specificity of FUT1 and FUT2 (Beyer et al., 1980Go; Kumazaki and Yoshida, 1984Go; Le Pendu et al., 1985Go; Betteridge and Watkins, 1985Go; Kyprianou et al., 1990Go; Larsen et al., 1990Go; Sarnesto, 1990Go, 1992; Kelly et al., 1995Go; Rouquier et al., 1995Go) and, as seen in Figure 4E, FUT1 can make H type 3 structures on PSGL-1/mIgG2b. Because we could not detect any H type 1 or 2 structures, but only H type 3 structures on PSGL-1/mIgG2b made in CHO cells following transfection with the H gene alone or in combination with the A gene (Figure 3C–E), and that these cells have been reported not to extend core 1 structures (Yeh et al., 2001Go), we conclude that the A and H epitopes are substitutions on core 1 O-linked glycans. This indicates that FUT1, as previously suggested in transfections of COS cells, can use core 1 precursors on glycoproteins (Liu et al., 1999Go).

Our results from western blot analysis further show that the blood group A antigen density on recombinant PSGL-1/mIgG2b when made in CHO cells by a combined H and A gene expression is low. When the enzymes were expressed in 293T cells in the same way, the A epitope density on the PSGL-1/mIgG2b was higher than in CHO cells (Figure 5), reflecting that there are likely to be more lactosamine sequences available for the FUT1 enzyme in the former cell type. In spite of the presence of H type 2 precursor chains in COS cells (Figure 3D), the A epitope density on the PSGL-1/mIgG2b made in COS cells with FUT1 and the {alpha}1,3 GalNAcT, was lower than on PSGL-1/mIgG2b made in CHO cells with the same enzymes (Figure 5). A possible explanation for this may be that more H type 3 precursor chains are available in CHO cells transfected with the H gene.

With regard to the number of H type 3 chain epitopes expressed (Figures 1 and 3E), PSGL-1/mIgG2b produced in 293T cells showed a different staining pattern compared to PSGL-1/mIgG2b produced in COS cells. The low expression of H type 3 chain structures on PSGL-1/mIgG2b made in COS cells indicate low levels of the core 1 precursor chain, or that this chain has been blocked by the action of another enzyme, for example, a sialyltransferase. Only a structural characterization of the glycans carried by PSGL-1/mIgG2b can resolve this issue.

Organ Tx across the ABO barrier is characterized by AMR mediated by preformed or induced antibodies against the donor organ blood group (Porter, 1963Go; Sanchez-Urdazpal et al., 1993Go; Farges et al., 1995Go; Tanabe et al., 1998Go; Alkhunaizi et al., 1999Go). There are several methods described for the purpose of removing anti-ABO antibodies, such as cryofiltration (Tamaki et al., 1998Go), plasma exchange (Bensinger et al., 1987Go), double filtration PP (Ishikawa et al., 1998Go), and EIA (Rieben et al., 1995Go). We propose to use a recombinant PSGL-1/mIgG2b carrying A determinants as the absorber in an EIA setting. Our results show that approximately 20 pmoles of recombinant PSGL-1/mIgG2b (calculated on a molecular weight of 300 kDa), corresponding to 2 nmoles of A determinants (based on 100 A determinants per mole of PSGL-1/mIgG2b) (Wilkins et al., 1996Go; Aeed et al., 1998Go, 2001) made in CHO-K1 with FUT2 and {alpha}1,3 GalNAcT could absorb 60% of the A-PAA-reactive antibodies. A-PAA-MPG corresponding to approximately 164,000 pmol of A trisaccharides (calculated on 2 µmol/g) was needed to absorb the same amount of anti-A antibodies. The amount of nonspecific protein absorption for both compounds was also assessed, and the nonspecific absorption was almost fourfold higher with the PAA-MPG-based compounds (data not shown). We believe the relatively higher absorption efficacy of mucin-based absorbers may depend on (1) multivalent carbohydrate substitution, (2) close spacing of carbohydrate epitopes, and (3) a structural versatility of the core saccharide chains that carry the immunodominant determinant. However, only a detailed structural characterization can verify these explanations. Furthermore, changing the protein backbone, for example, by making a synthetic mucin-type protein constructed by optimized mucin tandem repeats, may improve the absorber (Silverman et al., 2001Go). Also, cells engineered to express different combinations of GalNAc:polypeptide transferases may improve absorption efficacy by optimising the O-glycan substitution density.

In conclusion, we have investigated the repertoire of blood group A and H epitopes carried on recombinant PSGL-1/mIgG2b produced in various host cells expressing FUT1 or FUT2 together with the A gene {alpha}1,3 GalNAcT. In accordance with previous studies FUT1 gave rise to mostly H type 2 structures, whereas FUT2 in the absence of type 1 chain precursors (-Galß3GlcNAc-), clearly preferred O-linked core 1 precursors and thus mainly made H type 3 structures. The A epitope density on the PSGL-1/mIgG2b was highest in the CHO made protein, which on a carbohydrate epitope molar basis was at least 80 times better than A-PAA-MPG in absorbing anti-A antibodies.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cell culture
COS-7 m6 cells (Seed, 1987Go), CHO-K1 (ATCC CCL-61), and the SV40 Large T antigen expressing 293 human embryonic kidney cell line (293T; kindly provided by B. Seed), were cultured in Dulbecco’s modified Eagle’s medium (GibcoBrl, Life Technologies, Paisley, Scotland), supplemented with 10% fetal bovine serum (GibcoBrl, Life Technologies), 25 µg/ml gentamycin sulfate (Sigma, St. Louis, MO) and 2 mM glutamine (GibcoBrl, Life Technologies). The cells were passaged every 2–4 days. The HH14 hybridoma (ATCC HB-9299; U.S. patent 4,857,639) were cultured in RPMI 1640 (GibcoBrl, Life Technologies), supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 100 µg/µl of streptomycin, and 2 mM glutamine.

Purification of HH14 antibodies
Supernatant was collected from cultured HH14 cells. Two liters of supernatant was affinity purified on a goat anti-mouse IgM (Sigma) column, using a Bio-Rad LP chromatograph (Bio-Rad, Hercules, CA). The bound proteins were eluted by 0.1 M glycine-Cl, pH 2.5, and the eluate was immediately neutralized using 1 M Tris–Cl, pH 7.5. The eluate was dialyzed against 1% phosphate-buffered saline (PBS) and lyophilized. Lyophilized proteins were dissolved in distilled H2O to a final concentration of 3 µg/µl, as measured by the BCA assay.

Construction of expression vectors
The human blood group A gene was polymerase chain reaction (PCR) amplified off cDNA made from total RNA isolated from the MKN-45 cell line, using 5'-cgc ggg aag ctt gcc gag acc aga cgc gga-3' as forward primer and 5'-cgc ggg cgg ccg ctc acg ggt tcc gga ccg c-3' as reverse primer. The amplified cDNA (A gene) was subcloned into the polylinker of CDM8 (Seed, 1987Go) using Hind III and Not I. The blood group H gene was PCR amplified in two pieces using a human tonsil stroma library (Holgersson and Seed, unpublished data) as template. An internal Sse I site was created by PCR using internal overlapping primers changing nucleotide 775 (GenBank accession no. M35531) to a C, creating a Sse I site of the Pst I site. The cDNA encoding the carboxy terminal of FUT1 was amplified using 5'-ggg gac tac ctg cag gtt atg cct cag cgc-3' as forward primer and 5'-cgc ggg gcg gcc gct tca agg ctt agc caa tgt-3' as reverse primer, cleaved by Sse I and Not I, and subcloned into the CDM8 expression vector digested with Pst I and Not I. The cDNA encoding the amino terminal of FUT1 was amplified using 5'-cgc ggg aag ctt acc atg tgg ctc cgg agc cat-3' as forward primer and 5'-cca gcg ctg agg cat aac ctg cag gta gtc-3' as reverse primer, cleaved by Hind III and Sse I, and subcloned into CDM8 carrying the carboxy terminal following Hind III and Sse I cleavage.

The Se gene was similarly PCR amplified from cDNA reversely transcribed from total RNA isolated from peripheral blood mononuclear cells donated by a blood group A2Le(a-b+)Se individual, using 5'-cgc ggg aag ctt acc atg ctg gtc gtt cag atg-3' as forward primer and 5'-cgc ggg cgg ccg ctt agt gct tga gta agg g-3' as reverse primer. The Se gene cDNA was subcloned into CDM8 using Hind III and Not I. Glycosyltransferase cDNAs were sequenced and the enzymatic activity they encoded checked by flow cytometric analysis of transiently transfected cells using blood group H and A–specific monoclonal antibodies. The PSGL-1/mIgG2b chimera was constructed as described before (Liu et al., 1997Go).

Transfections and production of secreted PSGL-1/mIgG2b chimeras
The transfection cocktail was prepared by mixing 39 µl of 20% glucose, 39 µg of plasmid DNA, 127 µl dH2O, and 15.2 µl 0.1M polyethylenimine (25 kDa; Aldrich, Milwaukee, WI) in 5-ml polystyrene tubes. In all transfection mixtures, 13 µg of the PSGL-1/mIgG2b plasmid was used. Thirteen micrograms of the plasmid for the different glycosyltransferases were added, and, when necessary, the CDM8 plasmid was added to reach a total of 39 µg of plasmid DNA. The mixtures were left in room temperature for 10 min before being added in 10 ml of culture medium to the cells, at approximately 70% confluency. After 7 days, cell supernatants were collected, debris spun down (1400 x g, 15 min) and NaN3 was added to a final concentration of 0.02% (w/v).

Purification of secreted PSGL-1/mIgG2b for SDS–PAGE and western blot analysis
PSGL-1/mIgG2b fusion proteins were purified from collected supernatants on 50 µl goat anti-mIgG agarose beads (100 µl slurry; Sigma) by rolling head over tail overnight at 4°C. The beads with fusion proteins were washed three times in PBS and used for subsequent analysis. Typically, the sample was dissolved in 50 µl of 2x reducing sample buffer and 10 µl of sample was loaded in each well.

PNGaseF treatment of affinity-purified PSGL-1/mIgG2b
A PNGaseF kit (Roche Diagnostics, Indianapolis, IN) was used for N-glycan deglycosylation. A slight modification of the protocol provided by the manufacturer was used. In 1.5-ml Eppendorf tubes, 20 µl of reaction buffer was mixed with purified PSGL-1/mIgG2b on agarose beads and boiled for 3 min. The mixture was spun down, and 10 µl of the supernatant was transferred to a new Eppendorf tube. Ten microliters of PNGaseF or, as a negative control, 10 µl of reaction buffer were added. The tubes were incubated for 1.5 h at 37°C. After incubation, 20 µl of 2x reducing sample buffer and 10 µl of H2O was added, and the samples were boiled for 3 min.

ELISA for determination of PSGL-1/mIgG2b concentration in supernatants
Ninety-six-well ELISA plates (Costar 3590, Corning, NY) were coated with 0.5 µg/well of affinity-purified goat anti-mIgG specific antibodies (Sigma) in 50 µl of 50 mM carbonate buffer, pH 9.6, for tw2o h in room temperature. After blocking o/n at 4°C with 300 µl 3% bovine serum albumin (BSA) in PBS with 0.05% Tween (PBS-T) and subsequent washing, 50 µl sample supernatant was added, serially diluted in culture medium. Following washing, the plates were incubated for 2 h with 50 µl of goat anti-mIgM-HRP (Sigma), diluted 1:10,000 in blocking buffer. For the development solution, one tablet of 3, 3', 5, 5'-tetramethylbenzidine (Sigma) was dissolved in 11 ml of 0.05 M citrate/phosphate buffer with 3 µl 30 % (w/v) H2O2. One hundred microliters of development solution was added. The reaction was stopped with 25 µl 2 M H2SO4. The plates were read at 450 and 540 nm in an automated microplate reader (Bio-Tek Instruments, Winooski, VT). As a standard, a dilution series of purified mIgG Fc fragments (Sigma) in culture medium was used in triplicate.

SDS–PAGE and western blotting
SDS–PAGE was run by the method of Laemmli (1970)Go with a 5% stacking gel and an 8% resolving gel, and separated proteins were electrophoretically blotted onto HybondTM-C extra membranes as described before (Liu et al., 1997Go). Following blocking overnight in Tris-buffered saline with 0.05% Tween-20 (TBS-T) with 3% BSA, the membranes were washed three times with TBS-T. They were then incubated for 1 h in room temperature with mouse anti-human blood group A all types (mIgM, Dako, Carpinteria, CA) or anti-human H type 1 (mIgG3, Signet; Dedham, MA), H type 2 (mIgM, Dako) or H type 3 (mIgM, hybridoma HH14, ATCC HB9299). All antibodies were diluted 1:200 in 3% BSA in TBS-T, except for the H type 3 antibody, which was diluted to a concentration of 1 µg/ml in 3% BSA in TBS-T. The membranes were washed three times with TBS-T before incubation for 1 h at room temperature with secondary horseradish peroxidase (HRP)–conjugated antibodies, goat anti-mIgM (Cappel, Durham, NC) or goat anti-mIgG3 (Serotec, Oxford, England) diluted 1:2000 in 3% BSA in TBS-T. Bound secondary antibodies were visualized by chemiluminescence using the ECL kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the instructions of the manufacturer. For detection of the PSGL-1/mIgG2b itself, HRP-labeled goat anti-mIgG (Sigma) was used at a dilution of 1:10,000 in 3% BSA in TBS-T as described, but without incubation with a secondary antibody.

Determination of the relative blood group A epitope density on PSGL-1/mIgG2b
Western blots were run as described. The membranes were visualized in a Fluor-S Max MultImager carrying a CCD camera operating at –35°C (BioRad). Using the volume tool in the analysis window of the Quantity One software (BioRad), the volume (sum of the intensities of the pixels within the volume boundary x pixel area) for the blood group A reactivity was divided by the volume for the mIgG reactivity for the PSGL-1/mIgG2b made in COS, CHO, and 293T cells. To compare the A epitope/mouse IgG ratios between PSGL-1/mIgG2b made in the different host cells, the ratios were normalized to the ratio obtained from the A substituted PSGL-1/mIgG2b made in COS cells transfected with the H and A genes.

Absorption of serum
Six hundred microliters slurry of goat anti-mIgG agarose beads (Sigma) was transferred into 1.5-ml Eppendorf microcentrifuge tubes. The beads were spun down by a quick spin at 400 x g. The supernatant was then removed, and the beads were washed once with 1 ml PBS, spun down again, and transferred to 180 ml of supernatant from CHO cells transfected with cDNAs encoding PSGL-1/mIgG2b, the Se and the A gene. Supernatants containing agarose beads were incubated head over tail o/n at 4°C. For collection, the beads were spun down at 400 x g, 15 min, at room temperature, and transferred to 1.5-ml Eppendorf microcentrifuge tubes. Washing was done three times with PBS. These beads are referred to as A mucin-beads. Six hundred microliters of anti-mIgG agarose beads were also prepared in the same manner, but they were used for dilution of the A mucin-beads to obtain a dilution series of the A mucin-beads as absorbents. These beads are referred to as goat anti-mIgG beads. The beads were aliquoted into 4-ml Ellerman tubes according to Table I. A-PAA-MPG (Syntesome, Münich, Germany) and B-PAA-MPG (Syntesome) were weighed and aliquoted into Ellerman tubes according to Table I, and thereafter washed once with PBS.


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Table I. Serial dilutions of absorbents used for absorption of anti-blood group A antibodies from pooled serum of blood group O individuals
 
Pooled serum from five patients typed blood group O was obtained from the Blood Bank at Huddinge University Hospital (Ethical permission, Dnr. 392/99, approval date 08/15/2000). Cell debris was removed from the serum by centrifugation at 14,000 rpm for 5 min in a Jouan A-14 microcentrifuge. The cleared serum was transferred to another tube and incubated in a waterbath at 56°C for 1 h to inactivate complement. The serum was stored in aliquots at –20°C until being used.

Five hundred microliters of serum were added to each tube and mixed with the beads for 4 h on a rolling table at 4°C. Following absorption, the beads were spun down and the absorbed serum transferred to new Ellerman tubes. The absorbed serum was stored at –20°C until further analysis.

To determine the amount of PSGL-1/mIgG2b on the A mucin-beads, a goat anti-mIgG Fc ELISA (see previous procedure) was run on the supernatant before and after incubation with agarose beads.

ELISA for quantification of anti-A antibodies
Ninety-six-well ELISA plates (Costar 3590, Corning) were coated for 2 h at room temperature with 0.05 µg of A-PAA-biotin (Syntesome) in 50 µl per well of 50 mM carbonate buffer, pH 9.6. Following blocking o/n at 4°C with 300 µl 3% BSA in PBS-T and subsequent washing, 50 µl of serum serially diluted in PBS were added and the plate was incubated at room temperature for 2 h. After washing, the incubation was done with 50 µl of mouse-anti-human IgA, G and M-HRP (Jackson, PA) diluted 1:10,000 in blocking buffer. Development and reading of the plates was done as described previously.

Determination of total protein concentration in serum
The total protein concentration in serum was determined before and after absorption of anti-A antibodies. The microtiter plate protocol of the BCA (bicinchoninic acid) Protein Assay Reagent (Pierce, Rockford, IL) was used according to the manufacturer’s instructions, and samples were run in duplicates or triplicates.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We are grateful to H. Clausen for helping us with advice on antibodies and for providing the MKN45 cell line and I. Belyanchikov from Syntesome GmbH for kindly giving us the B-tri-PAA-MPG. We would also like to thank J. Liu and Z. He at Clinical Immunology, Karolinska Institutet and Marianne Backlund at Blodcentralen, Södersjukhuset, Stockholm, for technical assistance and the staff at Blodcentralen, Huddinge University Hospital AB, for providing us with serum. This work was supported by the Swedish Medical Research Council (no. K99-06X-13031-01A) and Karolinska Institutet. J.L. and J.H. were supported by a PhD fellowship and a senior scientist fellowship, respectively, from the program Glycoconjugates in Biological Systems, financed by the Swedish Foundation for Strategic Research.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
AMR, antibody-mediated rejection; BSA, bovine serum albumin; CHO, Chinese hamster ovary; COS, ; EIA, extracorporeal immunoabsorption; ELISA, enzyme-linked immunosorbent assay; FT, fucosyltransferase; GalNAcT, N-acetylgalactosaminyltransferase; HRP, horseradish peroxidase; mIgG, mouse IgG; mIgM, mouse IgM; MPG, macroporous glass beads; PAA, poly[N-(2-hydroxyethyl)acrylamide]; PBS, phosphate buffered saline; PBS-T, PBS with 0.05% Tween; PCR, polymerase chain reaction; PNGaseF, peptide:N-glycosidase F; PP, plasmapheresis; PSGL-1, P-selectin glycoprotein ligand-1; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBS-T, Tris-buffered saline with 0.05% Tween-20; Tx, transplantation.


    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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