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Glycobiology Advance Access originally published online on September 1, 2004
Glycobiology 2005 15(2):109-118; doi:10.1093/glycob/cwh146
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Glycobiology vol. 15 no. 2 © Oxford University Press 2005; all rights reserved.

Anti-{alpha}-galactosyl antibodies recognizing epitopes terminating with {alpha}1,4-linked galactose: human natural and mouse monoclonal anti-NOR and anti-P1 antibodies

Maria Duk2, Grazyna Kusnierz-Alejska3, Elena Y. Korchagina4, Nicolai V. Bovin4, Stanislawa Bochenek5 and Elwira Lisowska1,2

2 Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolf Weigl St. 12, 53-114 Wroclaw, Poland; 3 Institute of Hematology and Blood Transfusion, 00-957 Warsaw, Poland; 4 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, v-437 Moscow, Russian Federation; and 5 Regional Center for Blood Transfusion, 50-345 Wroclaw, Poland

1 To whom correspondence should be addressed; e-mail: lisowska{at}iitd.pan.wroc.pl

Received on July 13, 2004; revised on August 30, 2004; accepted on August 30, 2004


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
The rare NOR erythrocytes, which are agglutinated by most human sera, contain unique glycosphingolipids (globoside elongation products) terminating with the sequence Gal{alpha}1-4GalNAcß1-3Gal- recognized by common natural human antibodies. Anti-NOR antibodies were isolated from several human sera by affinity procedures, and their specificity was tested by inhibition of antibody binding to NOR-tri-polyacrylamide (PAA) conjugate (ELISA) by the synthetic oligosaccharides, Gal{alpha}1-4GalNAcß1-3Gal (NOR-tri), Gal{alpha}1-4GalNAc (NOR-di), Gal{alpha}1-4Galß1-3Galß1-4Glc ((Gal)3Glc), and Gal{alpha}1-4Gal (P1-di). Two major types of subspecificity of anti-NOR antibodies were found. Type 1 antibodies were found to react strongly with (Gal)3Glc and NOR-tri and weakly with P1-di and NOR-di, which indicated specificity for the trisaccharide epitope Gal{alpha}1-4Gal/GalNAcß1-3Gal. Type 2 antibodies were specific to Gal{alpha}1-4GalNAc, because they were inhibited most strongly by NOR-tri and NOR-di and were not (or very weakly) inhibited by (Gal)3Glc and P1-di. Monoclonal anti-NOR antibodies were obtained by immunizing mice with NOR-tri-human serum albumin (HSA) conjugate and were found to have type 2 specificity. All anti-NOR antibodies reacted specifically with NOR glycolipids on thin-layer plates. The cross-reactivity of type 1 anti-NOR antibodies with Gal{alpha}1-4Gal drew attention to a possible antigenic relationship between NOR and blood group P system glycolipids. The latter glycolipids include Pk (Gal{alpha}1-4Galß1-4Glc-Cer) present in all normal erythrocytes and P1 (Gal{alpha}1-4Galß1-4GlcNAcß1-3Galß1-4Glc-Cer) present only in P1 erythrocytes. Sera of some P2 (P1-negative) persons contain natural anti-P1 antibodies. This prompted us to test the specificity of anti-P1 antibodies. Natural human anti-P1 isolated from serum of P2 individual and mouse monoclonal anti-P1 were best inhibited by Gal{alpha}1-4Galß1-4GlcNAc (P1-tri) and did not react with NOR-tri and NOR-di. Monoclonal anti-P1 bound to Pk and P1 glycolipids and not to NOR glycolipids. These results indicated an entirely different specificity of anti-NOR and anti-P1 antibodies. Human serum samples differed in the content of anti-{alpha}-galactosyl antibodies, including both types of anti-NOR. In the sera of some individuals, type 1 or type 2 anti-NOR antibodies dominated, and other samples contained mixtures of both types of anti-NOR. The biological significance of these new abundant anti-{alpha}-galactosyl antibodies still awaits elucidation.

Key words: anti-{alpha}-galactosyl / anti-NOR / anti-P1 / glycolipids / polyagglutination


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
Carbohydrate epitopes terminated with an {alpha}-linked galactose residue are less common in human than in animal tissues, but human serum contains various natural anti-{alpha}-galactosyl antibodies. The anti-Gal{alpha}1,3Gal xenoantibodies, which recognize antigen absent from human tissues but abundant in glycoproteins and glycolipids of mammals up to New World monkeys, have been most widely studied because they are a major cause of acute rejection of xenotransplants (reviewed by Galili, 2001Go). An inherited NOR polyagglutination, identified so far in one American (Harris et al., 1982Go) and one Polish family (Kusnierz-Alejska et al., 1999Go), indicated the common presence of novel anti {alpha}-galactosyl antibodies in human sera.

Recently we found that NOR erythrocytes contain {alpha}-galactosylated glycosphingolipids that are products of globoside elongation and have the following structures: Gal{alpha}1-4GalNAcß1-3Gal{alpha}1-4Galß1-4Glc-Cer (NOR1) and Gal{alpha}1-4GalNAcß1-3Gal{alpha}1-4GalNAcß1-3Gal{alpha}1-4Galß1-4Glc-Cer (NOR2) (Duk et al., 2001Go, 2003bGo). These glycolipids are specifically recognized by human anti-NOR antibodies (Duk et al., 2001Go). Although the NOR condition is rare and does not cause health problems, anti-NOR antibodies are common and their characterization seemed interesting. Using synthetic oligosaccharides we found that anti-NOR antibodies are highly reactive with Gal{alpha}1-4GalNAcß1-3Gal, the terminal sequence unique to NOR glycolipids and not found in normal human tissues. Anti-NOR antibodies also reacted weakly with the disaccharides Gal{alpha}1-4GalNAc and Gal{alpha}1-4Gal but did not react with Gal{alpha}1-3Gal (Duk et al., 2003aGo). The terminal Gal{alpha}1-4Gal-sequence is present in humans in glycosphingolipids related to the blood group P system, that is, Pk (Gal{alpha}1-4Galß1-4Glc-Cer) and P1 (Gal{alpha}1-4Galß1-4GlcNAcß1-3Galß1-4Glc-Cer) (Reid and Lomas-Francis, 1997Go). The Pk glycolipid is expressed in all normal erythrocytes. The P1 antigen is present in erythrocytes of some individuals (~80% of Caucasians), which gives rise to the P1 and P2 (P1-negative) blood group phenotypes. Some P2 individuals have natural anti-P1 antibodies in their serum. These antibodies (and recently mouse monoclonal anti-P1 antibodies) are used for serological identification of P1 or P2 phenotype of erythrocytes, but immunochemical data on their fine specificity are scarce. Moreover, the common presence of anti-Gal{alpha}1,4Gal antibodies in human serum was reported (Wieslander et al., 1990Go), but the relation of these antibodies to anti-NOR or anti-P1 is unknown.

The studies described in this article were focused on a comparison of the fine specificity of natural human anti-NOR and anti-P1 antibodies using a panel of synthetic oligosaccharides and erythrocyte glycolipids. We also obtained two highly specific mouse monoclonal anti-NOR antibodies (monoclonal antibodies nor87 and nor118) that together with mouse monoclonal anti-P1 antibody (commercially available), were included in our studies. The results showed that anti-NOR and anti-P1 are distinct antibodies. Moreover, human polyclonal anti-NOR was shown to be a heterogeneous group of antibodies with respect to subspecificity. Generally, our data indicated a complexity of human natural humoral response to glycans terminating with {alpha}-galactose residues.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Anti-{alpha}-galactosyl antibodies in human serum
When enzyme-linked immunosorbent assay (ELISA) plates coated with seven {alpha}-galactosyl-oligosaccharide conjugates (listed in Table I) were overlayed with diluted serum samples of individual persons, the binding of immunoglobulins to all conjugates was observed (Figure 1). However, the binding showed a different strength and pattern for each serum sample. In most samples the binding to Gal{alpha}1-4GalNAcß1-3Gal (NOR-tri)-HSA was accompanied by lower, similar, or even higher binding to other conjugates, and one sample was found (serum 5 in Figure 1) that contained a significant level of anti-NOR antibodies and only trace amounts of other anti-{alpha}-galactosyl antibodies. This indicated that individuals display quantitative and qualitative differences in humoral response to {alpha}-galactosylated carbohydrate antigens.


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  		Table I. Oligosaccharides and oligosaccharide conjugates used

 


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  		Fig. 1. Binding of antibodies from human sera to various {alpha}-galactosyl oligosaccharide conjugates. The 100 times diluted serum samples from eight individuals were applied to the ELISA plates coated with seven conjugates (0.25 µg/well).

 
The structural and antigenic relationship between NOR and blood group P system antigens raised the question whether the level of anti-NOR antibodies depends on the P1/P2 status of the person. The binding of Igs from the sera of 10 P1 and 10 P2 individuals to NOR-tri-HSA-coated ELISA plates showed differences in the level of anti-NOR antibodies within each group, but no significant difference was noted between the P1 and P2 phenotypes (Figure 2). Binding of Ig from the same sera to P1-tri-polyacrylamide (PAA)-coated plates was weaker than to NOR-tri-HSA in the tested sera from P1 individuals and part of the sera of P2 persons (Figure 2), which could have resulted from cross-reactivity of anti-NOR antibodies with P1 (Duk et al., 2003aGo). However, 4 of 10 blood group P2 serum samples showed similar or even higher Ig binding to P1-tri-PAA than to NOR-tri-HSA, which indicated the presence of anti-P1 antibodies. Only two samples, which showed the highest Ig binding to P1-tri-PAA (Figure 2), agglutinated P1 erythrocytes. Agglutinating anti-P1 sera are not so frequent; these two samples were selected by serological testing of a larger number of serum samples from P2 persons and were included in our experiment together with eight nonagglutinating samples. The results suggested that human anti-P1 antibodies are distinct from anti-NOR.



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  		Fig. 2. Comparison of binding of antibodies from 100 times diluted human serum samples from 10 P1 and 10 P2 persons to ELISA plates coated with NOR-tri-HSA and P1-tri-PAA. Other details as in Figure 1.

 
Purification of human anti-NOR antibodies
Anti-NOR antibodies were purified either by a previously described method of adsorption to glutaraldehyde-fixed NOR erythrocytes (Duk et al., 2001Go, 2003aGo) or by using the NOR-tri-HSA-Sepharose column (Figure 3). All or most anti-NOR antibodies were bound to this column, and only part of them could be eluted with 0.02 M galactose. Galactose at higher concentrations (up to 0.5 M) did not elute more antibodies. The fraction eluted with 0.02 M galactose showed binding in ELISA not only to NOR-tri-HSA but also to other conjugates (Figure 3). Therefore this step was used to remove small amounts of less specific anti-{alpha}-galactosyl antibodies that bound weakly to the column, probably on the basis of recognition of the terminal {alpha}-galactose residue. The remaining anti-NOR antibodies were strongly bound to the column and were eluted with 5 M guanidine. This fraction contained antibodies that specifically bound to NOR-tri-HSA and in some cases showed cross-reactivity with P1-tri-PAA only (Figure 3). As shown by double immunodiffusion, most antibodies were of the IgG class, with a smaller amount of IgM.



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  		Fig. 3. Two examples of the purification of anti-NOR antibodies on the NOR-tri-HSA-Sepharose 4B column. Antibodies were purified from sera 1 and 7 (Figure 1) and the fractions obtained were tested for Ig binding to oligosaccharide conjugates as shown in Figure 1. (A) 100 times diluted serum applied to the column; (B) buffer eluate from the column adjusted to the same dilution as the serum; (C) 0.02 M galactose eluate; (D) 5 M guanidine eluate; C and D were diluted to about 10 times smaller volume than A and B.

 
Fine specificity of two major types of anti-NOR antibodies
Anti-NOR antibodies were isolated from the sera of different persons, using NOR erythrocytes (elution with galactose) or the affinity column (elution with guanidine) method and tested by inhibition of their binding to NOR-tri-PAA-coated ELISA plates. Surprisingly, different patterns of inhibition were obtained for anti-NOR isolated from individual sera.

We reported previously that anti-NOR are inhibited strongly by NOR-tri and over 100 times less strongly by NOR-di (Gal{alpha}1-4GalNAc) and P1-di (Gal{alpha}1-4Gal), and that P1-di was slightly stronger inhibitor than NOR-di (Duk et al., 2003aGo). This indicated that antibodies did not see a difference between Gal and GalNAc residues at position 2 (or slightly preferred the Gal residue at this position) and that the third Gal residue substituted at C3 was essential for the epitope activity. To prove this directly, the oligosaccharide Gal{alpha}1-4Galß1-3Galß1-4Glc- sp ((Gal)3Glc), with a terminal sequence differing from that of NOR-tri only by the presence of Gal instead of GalNAc at position 2, was synthesized and used as an inhibitor. The new anti-NOR antibody, which showed a pattern of inhibition closely similar to that reported earlier, was strongly inhibited by this tetrasaccharide. The inhibition was even 2–3 times stronger than by NOR-tri, 700 times than by P1-di, and over 2000 times stronger than by NOR-di (Figure 4A). This result fully confirmed the assumed specificity (called type 1) of this kind of anti-NOR antibody.



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  		Fig. 4. Inhibition of type 1 and type 2 human anti-NOR antibodies and monoclonal antibody nor87 with oligosaccharides and galactose. Inhibitors are shown in the insert. Binding of antibody/inhibitor samples to NOR-tri-PAA coated ELISA plates (1 µg/well) was measured. (A) Antibodies purified from serum 5 (Figure 1) by elution from the affinity column with guanidine; (B) antibodies obtained from serum 9 (not included into Figure 1) by adsorption on NOR erythrocytes; (C) monoclonal antibody nor87. Antibody nor118 showed an inhibition pattern closely similar to that of nor87.

 
In another serum sample a quite different type of anti-NOR antibody specificity (called type 2) was found. This antibody was best inhibited by NOR-tri (however, at much higher concentrations compared with type 1 anti-NOR), only 2 times less strongly by NOR-di, and was not inhibited by (Gal)3Glc and P1-di at concentrations 25 and 50 times higher than that required for 50% inhibition by NOR-di (Figure 4B). This showed that such an antibody recognized the disaccharide epitope Gal{alpha}1-4GalNAc unique to NOR glycolipids. The third Gal residue had minor importance in this epitope, but the second GalNAc residue could not be replaced by Gal.

Most tested sera apparently contained a mixture of both types of anti-NOR antibodies at different proportions. In Figure 5, three examples of such antibodies are shown. All were totally inhibited by NOR-tri. However (Gal)3Glc evidently inhibited only a part of antibodies, from ~90% (Figure 5A, dominant type 1) to 40% (Figure 5C, prevalence of type 2). Moreover, the weaker inhibition by (Gal)3Glc was accompanied by a relatively stronger inhibition by NOR-di. The fourth example, in Figure 5D, shows antibodies eluted from the affinity column with 0.02 M galactose. They differed from the guanidine-eluted antibodies from the same serum (Figure 5C) by a less specific inhibition pattern and relatively strong inhibition by galactose. These galactose-eluted antibodies also bound to several {alpha}-galactosyl oligosaccharide-PAA conjugates. Most likely this fraction contained antibodies (one kind or mixture) binding to various epitopes in which the terminal {alpha}-Gal residue was the most essential component.



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  		Fig. 5. Examples of inhibition of anti-NOR antibodies isolated from different human sera. (A) Serum 7 (Figure 1), antibody eluted with guanidine from the affinity column; (B) serum 10 (not included into Figure 1), antibody isolated by adsorption on NOR erythrocytes; (C and D) serum 2 (Figure 1), antibody eluted with galactose (D) and guanidine (C) from the affinity column. Other details as in Figure 4.

 
Due to the scarce amounts, trisaccharide P1-tri was only occasionally used for inhibition, and this inhibition was close to that of P1-di (data not shown). None of the antibodies were inhibited by Gal{alpha}1-3Gal at a 5 mM concentration. Specificity of the antibodies did not depend on the method of purification. Occasionally, the antibodies were purified from the same serum by two methods (adsorption on NOR erythrocytes and affinity column) and both preparations gave similar inhibition patterns. Anti-NOR antibodies were also tested by inhibition of agglutination of papain-treated NOR erythrocytes, as described by Duk et al. (2003a)Go. The results of hemagglutination inhibition were in good agreement with those obtained by inhibition of binding in ELISA (data not shown).

Mouse monoclonal anti-NOR antibodies
Monoclonal anti-NOR antibodies were obtained by immunization of mice with NOR-tri-HSA. Two clones were selected (nor87 and nor118) that produced antibodies specifically agglutinating NOR erythrocytes and showing closely similar specificities. Both antibodies bound only to NOR-tri-HSA and NOR-tri-PAA and did not bind to other conjugates listed in Table I (data not shown). The inhibition pattern of both antibodies (shown in the example of the mAb nor87 in Figure 4C) indicated a type 2 specificity. The antibody was strongly inhibited by NOR-tri and NOR-di, concentrations of 0.34 µM and 0.6 µM of these inhibitors, respectively, being required for 50% inhibition of binding. The 50% inhibition by (Gal)3Glc and P1-di occurred at much higher concentrations, that is, 130 µM and 230 µM, respectively. No inhibition by Gal{alpha}1-3Gal was observed.

Natural human and mouse monoclonal anti-P1 antibodies
Human anti-P1 antibodies were obtained from those sera of P2 persons that showed the highest binding to P1-tri-PAA (Figure 2). The antibodies were isolated by adsorption on glutaraldehyde-fixed blood group ABO–compatible P1 erythrocytes and elution with 0.5 M galactose. The obtained antibodies were compared with commercial monoclonal anti-P1 antibody. Both antibodies were tested with all available conjugates (Table I) and showed specific binding to P1-tri-PAA (data not shown). The monoclonal anti-P1 antibody was strongly inhibited by P1-tri and over 300 times less strongly by P1-di. There was no inhibition by NOR-related oligosaccharides (Figure 6). Human anti-P1 antibodies were also inhibited by P1-tri, but a much higher concentration of this inhibitor was necessary than in the case of monoclonal anti-P1, and probably for this reason there was no inhibition by P1-di at the concentration used. Surprisingly, in contrast to monoclonal anti-P1, at least part of the human antibodies was inhibited by (Gal)3Glc (Figure 6). However, inhibition by (Gal)3Glc was not related to the presence of type 1 anti-NOR antibodies, because there was no inhibition by NOR-tri and NOR-di. These results suggest that human P2 sera contain antibodies recognizing different trisaccharide epitopes terminating with Gal{alpha}1-4Gal, but not those terminating with Gal{alpha}1-4GalNAc.



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  		Fig. 6. Inhibition of human and mouse monoclonal anti-P1 antibodies with oligosaccharides shown in the insert. Inhibition of binding to P1-tri-PAA coated ELISA plates (0.1 µg/well) was measured.

 
Reactivity of antibodies with neutral glycosphingolipids of human erythrocytes
The total neutral glycolipid fractions isolated from NOR and control erythrocytes were fractionated by high-performance thin-layer chromatography (HPTLC), and the plates were overlayed with antibodies to detect reactive glycolipids. All tested preparations of human anti-NOR antibodies and monoclonal anti-NOR antibodies reacted with NOR1 and NOR2 glycolipids of NOR erythrocytes and did not detect any glycolipids in control erythrocytes (see examples in Figure 7). A characteristic difference in binding of type 1 and type 2 anti-NOR antibodies was observed while staining the plates. In the case of type 1 antibodies, the NOR1 band appeared faster and was more intensely stained than the double band in the NOR2 region (Figure 7, lanes 1 and 2). In contrast, human anti-NOR containing only type 2 antibodies stained the NOR2 glycolipid only (Figure 7, lane 3). The monoclonal antibody nor87 (type 2) showed a similar tendency: The NOR2 band appeared first and was strongly stained, but a weaker NOR1 band became visible after prolonged incubation with the substrate (Figure 7, lane 4).



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  		Fig. 7. Binding of anti-NOR antibodies to human erythrocyte neutral glycolipids fractionated by HTPLC. Lanes A and NOR, glycolipids from control blood group A and NOR erythrocytes, respectively. Orcinol staining shows the presence of NOR1 and NORint glycolipids in NOR erythrocytes; the NOR2 band is not detected by orcinol staining. NORint is an intermediate product between NOR1 and NOR2, terminating with GalNAc and not reactive with anti-NOR antibodies (Duk et al., 2003bGo). The following anti-NOR antibodies were used: 1 and 2, antibodies with type 1 specificity isolated from sera 5 and 7, respectively; 3 and 4, antibodies with type 2 specificity, human from serum 9, and mAb nor87, respectively. Inhibition of these antibodies is shown in Figures 4 and 5.

 
The monoclonal anti-P1 antibody was similarly tested with glycolipids from NOR (P1) and control P1 and P2 erythrocytes. It detected the Pk glycolipid in all erythrocytes and the P1 glycolipid band (migrating slightly more slowely than NOR1) in P1 erythrocytes only (Figure 8). The isolated human anti-P1 antibodies were too weak, or their amount was too small, to perform this assay.



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  		Fig. 8. Binding of mouse monoclonal anti-P1 antibody to human erythrocyte neutral glycolipids isolated from normal P1 and P2 erythrocytes and NOR (P1) erythrocytes. To compare the positions of the Pk and P1 bands with those of NOR1 and NOR2, staining of the same glycolipid samples with the mAb nor118 is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
The rare NOR erythrocytes, which are agglutinated by most human sera, contain unique glycolipids terminating with the sequence Gal{alpha}1-4GalNAcß1-3Gal-. The results described herein showed that the terminal sequence of NOR glycosphingolipids is recognized by two major types of natural human antibodies. The epitopes recognized by these antibodies can be presented as follows:


Anti-NOR antibodies of type 1 strongly reacted with Gal{alpha}1-4GalNAcß1-3Gal- and Gal{alpha}1-4Galß1-3Gal-, where the presence of the third Gal residue was essential and the lack of the acetyl-amino group on the GalNAc residue even slightly increased the reactivity. It is possible that anti-NOR type 1 antibodies were detected as anti-Gal{alpha}1-4Gal antibodies by Wieslander et al. (1990)Go. Antibodies that do not see an essential difference between Gal and GalNAc are known. For example, blood group O patients who underwent transplantation with either A or B kidney produced an antibody that reacted with blood group A and B epitopes and also with Gal{alpha}–bovine serum albumin conjugate (Galili et al., 2002Go). Anti-NOR antibodies with type 2 specificity evidently recognized the Gal{alpha}1-4GalNAc sequence, the GalNAc residue could not be replaced by Gal, and addition of the third Gal residue, in contrast to type 1 specificity, had a minor effect on the reaction. Similar (type 2) specificity was shown by murine monoclonal anti-NOR antibodies nor87 and nor118. The obtained monoclonal antibodies that react strongly with Gal{alpha}1-4GalNAc and several hundred times less strongly with Gal{alpha}1-4Gal are good potential reagents for looking for a unique Gal{alpha}1-4GalNAc sequence in biological material.

Both types of anti-NOR antibodies reacted specifically with NOR-related glycolipids, NOR1 and NOR2. These two glycolipids represent globoside elongated by a HGal{alpha}1-4 (NOR1) or Gal{alpha}1-4GalNAß1-3Gal{alpha}1-4 (NOR2) unit (Duk et al., 2001Go, 2003bGo). Orcinol staining of neutral glycolipids from NOR erythrocytes suggests that the content of NOR1 is higher than that of NOR2 glycolipid, because the NOR1 band is distinctly stained, but no band unique for the NOR phenotype is seen in the NOR2 region (Figure 7). Despite this, however, type 2 human anti-NOR antibodies reacted only with NOR2 glycolipid, and monoclonal antibody nor87 reacted more strongly with NOR2 than with NOR1 glycolipid. It is possible that type 2 human anti-NOR are in fact specific to an epitope containing two consecutive NOR-di units, and such epitope is present only in the NOR2 glycolipid. This may explain why human type 2 anti-NOR antibodies required relatively high concentrations of NOR-di and NOR-tri oligosaccharides for inhibition (Figure 4). On the other hand, the epitope with the double NOR-di unit may have only moderately higher reactivity with monoclonal anti-NOR antibodies than does the single NOR-di unit. This was indicated by a strong inhibiton of monoclonal antibodies by NOR-tri and NOR-di and by the reactivity of these antibodies with NOR1 and NOR2 glycolipids. A stronger reaction with oligomers of Gal{alpha}1-3Gal than with monomers has been shown for anti-Gal{alpha}1,3Gal IgG antibodies (Rieben et al., 2000Go). Otherwise, the distance of the epitope from the ceramide (length of the oligosaccharide chain) may also play a role in the reaction of anti-NOR antibodies with glycolipids.

The specificity of the antibodies reacting with the NOR-tri affinity column and eluted with 0.02 M galactose indicated that human sera contain natural antibodies binding to various {alpha}-Gal-terminated epitopes in which further carbohydrate components have relatively lower significance, similarly as in the case of elicited antibodies described by Galili et al. (2002)Go. Our results indicated differences in the content of anti-NOR antibodies in human sera. The sera differed in the levels of the antibodies, similar to what was found for anti-Gal{alpha}1,3Gal antibodies (Buonomano et al., 1999Go; Lisowska and Duk, 2004Go). Moreover, individual sera differed in the proportion of less specific NOR-reactive antibodies eluted with galactose from the affinity column and highly specific anti-NOR antibodies with type 1 and type 2 specificity. Our experience, based on testing antibodies from different individual sera. suggests that anti-NOR with type 1 specificity are more abundant and common than type 2 antibodies.

Despite the cross-reactivity of type 1 anti-NOR with Gal{alpha}1-4Gal (the terminal sequence of P1 and Pk antigens), human and mouse monoclonal anti-P1 antibodies showed quite different specificity. They were inhibited by the P1-tri (terminal trisaccharide sequence of the P1 glycolipid) and did not react with NOR-related oligosaccharides. This indicated that the second Gal residue in the P1 epitope cannot be replaced by GalNAc. The monoclonal anti-P1, when tested with glycolipids on the HPTLC plate, reacted most strongly with the Pk glycolipid present in all erythrocytes, and detected the P1 glycolipid in P1 erythrocytes. The serological anti-P1 specificity of this antibody results most likely from the fact that the commonly present Pk glycolipid is too short to be available for the antibody in the erythrocyte membrane. The P1/P2 blood group system is still a puzzle, because specific {alpha}1,4-galactosyltransferase synthesizing the P1 antigen has not been cloned. Moreover, the generally low expression of the P1 antigen in P1 erythrocytes shows exceptionally high heterogeneity seen in hemagglutination, so that the difference between P1 and P2 cells seems to be rather quantitative than qualitative. A recent paper by Iwamura et al. (2003)Go shed some light on this problem. The authors, in contrast to earlier findings, obtained data indicating that P1 and Pk antigens are synthesized by the same earlier cloned Pk synthase and that erythrocytes serologically typed as P2 contained P1 antigen, although its amounts were lower than in P1 cells. Iwamura et al. (2003)Go suggested that transcriptional regulation of Pk synthase may be a major factor determining the P1/P2 phenotype. This may explain why not all P2 sera contain anti-P1 alloantibodies. Perhaps the antibodies are formed only in persons with null or extremely low expression of P1 glycolipid.

In conclusion, our results add new information on a complexity of natural anti-{alpha}-galactosyl antibodies in human sera. In addition to the widely studied anti-Gal{alpha}1,3Gal and related anti-{alpha}-galactosyl antibodies, human sera contain a similarly abundant group of natural antibodies recognizing epitopes terminating with {alpha}1,4-linked galactose. These antibodies, reactive with glycolipids responsible for NOR polyagglutination, display at least two major subspecificities and are distinct from anti-P1 antibodies occurring in sera of P2 individuals. Looking for the role of anti-Gal{alpha}1,3Gal antibodies (besides the rejection of xenotransplants) showed that their level is increased under various pathological conditions and their contribution to the pathogenesis of thyroid gland disorders and other diseases has been hypothesized (D'Alessandro et al., 2002Go; Knobel et al., 1999Go; Winand et al., 1994Go). Further studies are necessary to find the biological significance of the anti-NOR antibodies.


   Materials and methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Human erythrocytes and sera
The serologically typed human erythrocyte and serum samples were obtained from the Regional Transfusion Center in Wroclaw or from the Institute of Hematology and Transfusiology in Warsaw. The NOR blood was donated by our NOR-positive donor ST of blood group A2, P1.

Oligosaccharides and oligosaccharide conjugates
The NOR-related oligosaccharides (NOR-di and NOR-tri) were obtained by chemical synthesis (Westerlind et al., 2002Go). The NOR-tri-HSA and NOR-tri-PAA conjugates were obtained by using the N-acryloyl derivative of NOR-tri (prepared as described by Kallin et al., 1989Go) for copolymerization with HSA or PAA, according to the procedures reported by Romanowska et al. (1994)Go and Kallin (1994)Go, respectively. Analyses of NOR-tri-HSA and NOR-tri-PAA by matrix-assisted laser desorption ionization time-of-flight spectroscopy and 1H-nuclear magnetic resonance (NMR), respectively, indicated an average substitution degree of 23 trisaccharides per HSA molecule and 1 trisaccharide per 13 acrylamide residues. The P1-tri and the oligosaccharide-PAA conjugates (of Mr around 30,000 Da, containing ~20% sugars) were synthesized by described methods (Bovin, 1998Go). The disaccharides P1-di and Gal{alpha}1-3Gal were purchased from Glycorex (Lund, Sweden).

Synthesis of 3-trifluoroacetamidopropyl Gal{alpha}1-4Galß1-3Galß1-4Glcß (1)
A mixture of 3-azidopropyl O-(2,6-di-O-benzyl-ß-D-galactopyranosyl)-(1->4)-2,3,6-tri-O-benzyl-ß-D-glucopyranoside (obtained as described by Siebert et al., 2003Go) (185 mg, 0.211 mmole), silver trifluoromethanesulfonate (36 mg, 0.140 mmole), 1,1,3,3-tetramethylurea (17 ml, 0.142 mmole) and molecular sieves (4 Å) in dry dichloromethane (5 ml) was stirred at 20°C for 30 min. Then a solution of O-(2,3,4,6-tetra-O-acetyl-{alpha}-D-galactopyranosyl)-(1->4)-2,3,6-tri-O-acetyl-{alpha}-D-galactopyranosyl bromide (prepared from 95 mg [0.140 mmole] of [2,3,4,6-tetra-O-acetyl-{alpha}-D-galactopyranosyl]-[1->4]-1,2,3,6-tetra-O-acetyl-{alpha}/ß-D-galactopyranose, as described by Davis et al., 2003Go) in dry dichloromethane (5 ml) was added dropwise. After stirring for 18 h at 20°C the mixture was filtered through Celite and concentrated in vacuo. The crude mixture was O-acetylated with acetic anhydride in pyridine and concentrated in vacuo. Column chromatography (Silica gel 60, Merck, Darmstadt, Germany, toluene/ethyl acetate, 7:1 -> 1:1) of the residue yielded 3-azidopropyl O-(2,3,4,6-tetra-O-acetyl-{alpha}-D-galactopyranosyl)-(1->4)-(2,3,6-tri-O-acetyl-ß-D-galactopyranosyl)-(1->3)-(4-O-acetyl-2,6-di-O-benzyl-ß-D-galacto-pyranosyl)-(1->4)-2,3,6-tri-O-benzyl-ß-D-glucopyranoside (2) (67 mg, 31%) as a colorless foam. Thin-layer chromatography (foil plates, silica gel 60 F254, Merck, toluene/ethyl acetate, 2:1): Rf = 0.35. 1H-NMR (Bruker WM 500, 500 MHz, internal standard tetramethylsilane, CDCl3): 1.89 (m, 2H, CH2), 1.75–2.14 (8 s, 24H, 8Ac), 3.31–5.63 [m, 42H, H,H-correlation spectroscopy: 3.31 (H-5a), 3.36 (H-2a), 3.39 (CH2NH), 3.54 (H-3a), 3.67 (H-3b), 3.77 (H-4a), 4.04 (H-4c), 4.34 (d, J1,2 = 7.58 Hz, H-1a), 4.48 (d, J1,2 = 7.83 Hz, H-1b), 4.77 (d, J1,2 = 7.58 Hz, H-1c), 4.81 (H-3c), 5.01 (d, J1,2 = 3.42 Hz, H-1d), 5.17 (dd, J1,2 = 7.58 Hz, J2,3 = 10.76 Hz, H-2c), 5.22(dd, J1,2 = 3.42 Hz, J2,3 = 11.01Hz, H-2d), 5.42 (br.d, J3,4 = 3.42Hz, H-4b), 5.52 (dd, J3,2 = 11.01 Hz, J3,4 = 3.18 Hz, H-3d), 5.63 (br.d, J3,4 = 3.18 Hz, H-4d)], 7.14–7.42 (m, 30H, 5Ph). Unprotected tetrasaccharide 1 was obtained from 2 (67 mg) by reduction of the azido group with triphenyl phosphine in aqueous tetrahydrofuran (Vaultier et al., 1983Go), followed by N-trifluoroacetylation with methyl trifluoroacetate in methanol/triethylamine, O-deacetylation in methanolic NaOMe, and catalytic hydrogenolysis (Pd/C). Tetrasaccharide 1 was obtained with the yield of 67% (24 mg). Thin-layer chromatography: Rf = 0.26 (ethanol/chloroform/water, 6:3:1). 1H-NMR (D2O): 1.94 (m, 2H, CH2), 3.34 (m, 1-H, H-5a), 3.46 (m, 2H, CH2-N), 4.05 (m, 2H, H-5b, H-5d), 4.17 (br.d, J4,3 = 3.5, H-4c), 4.38 (m, 1-H, H-5c), 4.5 (d, 1-H, J1,2 = 7.9 Hz, H-1a), 4.52 (d, 1-H, J1,2 = 7.8 Hz, H-1b), 4.72 (d, 1-H, J1,2 = 7.6 Hz, H-1c), 4.98 (d, 1-H, J1,2 = 4 Hz, H-1d).

Monoclonal antibodies
Monoclonal anti-NOR antibodies were obtained by immunization of BALB/c mice with NOR-tri-HSA. The mice were injected subcutaneously with 100 µg NOR-tri-HSA emulsified in complete Freund's adjuvant. Second and third injection (50 µg, intraperitoneally) at 14-day intervals were given after complete disappearance of subcutaneous inflammatory granuloma, and booster dose (10 µg, intravenously) was given 3 days before fusion. Spleen cells of immunized mice were fused with SP-2/O cells and subsequently subcloned by limiting dilutions. Screening for positive clones was done by binding to NOR-tri-PAA (ELISA) and by hemagglutination of papain-treated NOR erythrocytes. Two sample colonies were selected and established as clones (nor87 and nor118) and were expanded in vitro. Antibodies (both IgG1) were used as diluted cell culture supernatants. Anti-P1 monoclonal antibody was purchased from DIAGAST (Loos, France).

Purification of human anti-NOR and anti-P1 antibodies
Anti-NOR and anti-P1 antibodies were isolated from blood group ABO–compatible human sera by adsorption on glutaraldehyde-fixed NOR or normal P1 erythrocytes, respectively, and elution with 0.5 M galactose (Duk et al., 2001Go). Anti-NOR antibodies were obtained from sera of P1 individuals (to avoid adsorption of anti-P1 antibodies), and anti-P1 antibodies were isolated from those sera of P2 persons which agglutinated P1 erythrocytes.

Alternatively, anti-NOR antibodies were isolated by affinity chromatography on the NOR-tri-HSA-Sepharose 4B column. NOR-tri-HSA was bound to cyanogen bromide–activated Sepharose 4B (Pharmacia, Uppsala, Sweden) by the routine method; the adsorbent obtained contained 1 mg NOR-tri-HSA per milliliter. The serum was filtered through a Syringer polyvinylidene difluoride (0.45 µm) filter (Millipore, Bedford, MA). About 10 ml of filtered serum was diluted 3 times with phosphate buffered saline (PBS) (10 mM phosphate buffer/0.15 M NaCl, pH 7.4, containing 0.02% NaN3) and applied to 1 ml affinity column. The column was washed with PBS until unbound proteins were eluted and then with 0.02 M galactose in the buffer; elution of proteins was checked by reading A280 in 400 µl fractions. The protein-containing fractions were pooled and passed through a PD-10 column (Pharmacia) in PBS (to remove galactose). This eluate was found to contain immunoglobulins that bound to several oligosaccharide–conjugates (ELISA), including NOR-tri-HSA. Most of the anti-NOR antibodies were strongly bound to the column and were not eluted at higher galactose concentrations, up to 0.5 M. They were eluted most efficiently with 5 M guanidine hydrochloride in PBS, the guanidine was quickly removed using the PD-10 column, and this fraction, containing purified anti-NOR antibodies, was used for further testing.

Double immunodiffusion
Ig class of monoclonal and human anti-NOR antibodies was determined by double immunodiffusion in agarose gel (Ouchterlony, 1958Go). Specific antibodies against mouse IgG1, IgG2a, IgG2b, IgM, and IgA were from Sigma, and those against human IgG, IgA, and IgM were from Dako (Glostrup, Denmark).

ELISA of antibody binding and inhibition of binding
Generally, the previously described procedures were used (Duk et al., 2003aGo). Briefly, the ELISA plates (Nunc, MaxiSorp, Roeskilde, Denmark) were coated with oligosaccharide conjugates at concentrations indicated in the legends to the figures. The serum (filtered through Syringer filters) or antibody samples were diluted with 0.05 M Tris–HCl buffer, pH 7.4/0.15 M NaCl containing 0.05% Tween 20 and 1% HSA (dilutions of other reagents were done with the buffer containing 0.1% HSA). In the case of human antibodies, the coated plates were consecutively incubated with the antibody tested (1 h), biotinylated goat antibodies against human Ig (Sigma) (1 h), ExtrAvidin–alkaline phosphatase conjugate (Sigma) (1 h) and the substrate p-nitrophenyl phosphate (Sigma 104 Phosphatase Substrate tablets) until color developed, usually 30–60 min. For mouse monoclonal antibodies, alkaline phosphatase–conjugated goat antibodies against mouse Ig (Dako) were used. The adsorbance was read at 405 nm in an ELISA reader.

The reactivity of antibodies with oligosaccharides was measured by inhibition of binding of anti-NOR and anti-P1 antibodies to NOR-tri-PAA and P1-tri-PAA, respectively. Antibody samples at constant dilution (giving after twofold dilution with buffer a control binding value with A405 absorbance of around 0.5) were mixed with an equal volume of serially diluted inhibitor (or buffer for control samples), incubated for 1 h, and used for the binding assay. The binding was performed as described, except that the incubation time of antibody-inhibitor samples on the plates was shortened to 30 min to increase the sensitivity of the assay. The results are shown as percent of inhibition of the control binding.

All tests were performed in duplicate (the difference between parallel samples usually did not exceed 3%), the binding to buffer-coated wells (negligible for purified antibodies and slightly higher for sera) was subtracted, and the mean values are given. Other details were as reported earlier (Duk et al., 2003aGo).

Binding of antibodies to human erythrocyte neutral glycolipids on HPTLC plates
The isolation of erythrocyte neutral glycolipids, HPTLC on Kieselgel 60 (Merck) plates and the detection of glycolipids on the plates with antibodies were as described (Duk et al., 2001Go; Kusnierz-Alejska et al., 1999Go). Total neutral glycolipid fraction isolated from 50 ml of packed erythrocytes was dissolved in 1.3 ml of chloroform/methanol (2:1), and 4 µl of this solution was applied on each lane of HPTLC plates. The binding of antibodies to glycolipids was detected with the same reagents as in the ELISA assay, except that nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma) was used as a substrate.


   Acknowledgements
 
The authors thank Drs. Iga Steuden and Jerzy Szkudlarek (Middle-European Diagnostics, Wroclaw) for obtaining hybridoma clones producing monoclonal anti-NOR antibodies. This work was supported by the grant no. 3PO5A 109 24 (to M.D., G.K.-A., S.B., and E.L.) of the State Committee for Scientific Research, Warsaw.


   Abbreviations
 
ELISA, enzyme-linked immunosorbent assay; HPTLC, high-performance thin-layer chromatography; HSA, human serum albumis; NMR, nuclear magnetic resonance; NOR1, Gal{alpha}1-4GalNAcß1-3Gal{alpha}1-4Galß1-4Glc-Cer; NOR2, Gal{alpha}1-4GalNAcß1-3Gal{alpha}1-4GalNAcß1-3Gal{alpha}1-4Galß1-4Glc-Cer; P1, Gal{alpha}1-4Galß1-4GlcNAcß1-3Galß1-4Glc-Cer; PAA, polyacrylamide; PBS, phosphate buffered saline; Pk, Gal{alpha}1-4Galß1-4Glc-Cer


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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M. Duk, S. Singh, V. N. Reinhold, H. Krotkiewski, E. Kurowska, and E. Lisowska
Structures of unique globoside elongation products present in erythrocytes with a rare NOR phenotype
Glycobiology, March 1, 2007; 17(3): 304 - 312.
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