Studies on Gal{alpha}3-binding proteins: comparison of the glycosphingolipid binding specificities of Marasmius oreades lectin and Euonymus europaeus lectin

Susann Teneberg1,2, Björn Alsén2, Jonas Ångström2, Harry C. Winter3 and Irwin J. Goldstein3

2 Institute of Medical Biochemistry, Göteborg University, P.O. Box 440, SE 405 30 Göteborg, Sweden
3 Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606, USA

Received on December 6, 2002; revised on January 16, 2003; accepted on January 21, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The carbohydrate binding preferences of the Gal{alpha}3Galß4 GlcNAc-binding lectins from Marasmius oreades and Euonymus europaeus were examined by binding to glycosphingolipids on thin-layer chromatograms and in microtiter wells. The M. oreades lectin bound to Gal{alpha}3-terminated glycosphingolipids with a preference for type 2 chains. The B6 type 2 glycosphingolipid (Gal{alpha}3[Fuc{alpha}2]Galß4GlcNAcß3Galß4Glcß1Cer) was preferred over the B5 glycosphingolipid (Gal{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer), suggesting that the {alpha}2-linked Fuc is accommodated in the carbohydrate binding site, providing additional interactions. The lectin from E. europaeus had broader binding specificity. The B6 type 2 glycosphingolipid was the best ligand also for this lectin, but binding to the B6 type 1 glycosphingolipid (Gal{alpha}3[Fuc{alpha}2]Galß3GlcNAcß3Galß4Glcß1Cer) was also obtained. Furthermore, the H5 type 2 glycosphingolipid (Fuc{alpha}2Galß4GlcNAcß3Galß4Glcß1Cer), devoid of a terminal {alpha}3-linked Gal, was preferred over the the B5 glycosphingolipid, demonstrating a significant contribution to the binding affinity by the {alpha}2-linked Fuc. The more tolerant nature of the lectin from E. europaeus was also demonstrated by the binding of this lectin, but not the M. oreades lectin, to the x2 glycosphingolipid (GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer) and GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. The A6 type 2 glycosphingolipid (GalNAc{alpha}3[Fuc{alpha}2]Galß4GlcNAcß3Galß4Glcß1Cer) and GalNAc{alpha}3Galß4GlcNAcß3Galß4Glcß1-Cer were not recognized by the lectins despite the interaction with B6 type 2 glycosphingolipid and the B5 glycosphingolipid. These observations are explained by the absolute requirement of a free hydroxyl in the 2-position of Gal{alpha}3 and that the E. europaea lectin can accommodate a GlcNAc acetamido moiety close to this position by reorienting the terminal sugar, whereas the M. oreades lectin cannot.

Key words: Euonymus europaeus lectin / glycosphingolipid binding / Gal{alpha}3Gal epitope / Marasmius oreades lectin


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The Gal{alpha}3Gal and Gal{alpha}3Galß4GlcNAc carbohydrate epitopes are of common occurrence in many biological systems. These include the following: basement membranes of rodents and other nonprimate mammals (Peters and Goldstein, 1979Go; Okada and Spiro, 1980Go; Edge and Spiro, 1987Go); 3T3 (Stanley et al., 1979Go) and Ehrlich ascites tumor cells (Eckhardt and Goldstein, 1983Go); bovine thyroglobulin (Okada and Spiro, 1980Go; Spiro and Boyroo, 1984); porcine tissues and organs (Sandrin and Mckenzie, 1994Go); and glycoconjugates from New World primates but not Old World monkeys, apes, and humans (Galili, 1999Go). Human blood group B erythrocytes, however, contain this epitope with an additional {alpha}2-linked fucose residue (Gal{alpha}3[Fuc{alpha}2]Gal). Several carbohydrate-binding proteins recognize and bind to the Gal{alpha}3Gal- and Gal{alpha}3Galß4GlcNAc-saccharides. A previous report delineated recognition of glycosphingolipids containing these carbohydrate epitopes by three proteins: toxin A of Clostridium difficile, human natural {alpha}-galactosyl IgG, and the monoclonal antibody Gal-13. Interestingly, all three proteins also bound to GalNAc{alpha}/ß3Galß4GlcNAc- and GlcNAcß3Galß4GlcNAc-terminated glycosphingolipids but failed to bind to type 1 or type 2 branched blood group B glycosphingolipids terminating in Gal{alpha}3(Fuc{alpha}2) Gal- end groups (Teneberg et al., 1996Go).

A recent communication described the isolation, characterization, and carbohydrate-binding properties of the lectin present in the mushroom Marasmius oreades (Winter et al., 2002Go). This lectin recognizes the Gal{alpha}3Galß4GlcNAc epitope but also binds to the branched blood group B epitope noted in the previous paragraph. The Gal{alpha}3Galß4- GlcNAc epitope and the blood group B epitope are also recognized by the lectin from Euonymus europaeus, which in addition binds to the blood group O epitope (Fuc{alpha}2Gal-) (Petryniak and Goldstein, 1986Go, 1987Go). In this study the binding of the mushroom lectin and the E. europaeus lectin (EEA) to a large series of glycosphingolipids was investigated to compare them with the three cross-binding Gal{alpha}3Galß4GlcNAc-binding proteins studied previously.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Chromatogram binding assays
The initial screening for M. oreades lectin (MOA) carbohydrate recognition using mixtures of glycosphingolipids from various sources separated on thin-layer plates revealed a selective interaction with fractions containing Gal{alpha}3- terminated compounds. As shown in Figure 1B, the lectin bound to a number of slow-migrating compounds in the nonacid glycosphingolipid fraction of human blood group B erythrocytes (lane 2) and also to the fractions of human blood group AB erythrocytes (lane 1) and horse erythrocytes (lane 4), whereas no binding to the nonacid fractions of human blood group O erythrocytes (lane 3) or blood group A erythrocytes (not shown) occurred.



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Fig. 1. Binding of 125I-labeled Marasmius oreades lectin and Euonymus europaeus lectin to mixtures of glycosphingolipids on thin-layer chromatograms. Chemical detection by anisaldehyde (A) and autoradiograms obtained by binding of 125I-labeled MOA (B) and EEA (C). The glycosphingolipids were separated on aluminum-backed silica gel plates, using chloroform/methanol/water (60:35:8 v/v/v) as solvent system, and the binding assays were performed as described under Materials and methods. The lanes were as follows: lane 1, nonacid glycosphingolipids of human blood group AB erythrocytes, 40 µg; lane 2, nonacid glycosphingolipids of human blood group B erythrocytes, 40 µg; lane 3, nonacid glycosphingolipids of human blood group O erythrocytes, 40 µg; lane 4, nonacid glycosphingolipids of horse erythrocytes, 40 µg; lane 5, Gal{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer (B5 pentaglycosylceramide), 4 µg. Autoradiography was for 12 h.

 
The EEA (Figure 1C) also bound to slow-migrating nonacid glycosphingolipids of human blood group AB erythrocytes (lane 1), human blood group B erythrocytes (lane 2), and horse erythrocytes (lane 4). In addition, this lectin interacted with slow-migrating glycosphingolipids in the nonacid fraction of human blood group O erythrocytes (lane 3), but no binding to the nonacid fraction of blood group A erythrocytes was obtained (not shown). A further notation is that the major compounds of the human erythrocyte nonacid glycosphingolpid fractions, that is, the Gal{alpha}4Gal-containing globotriaosylceramide and globoside, were not recognized by either lectin.

Further binding assays were done using pure glycosphingolipids. The results are exemplified in Figures 2Go4 and summarized in Table I. Due to a heterogenous ceramide composition, the pure glycosphingolipids are occasionally separated into several bands on the thin-layer chromatograms. With the exception of isoglobotriaosylceramide (Gal{alpha}3Galß4Glcß1Cer; Figure 2B, lane 1), the MOA bound to all glycosphingolipids with terminal Gal{alpha}3 (nos. 16, 17, 24, 25, 28, 30, 40, and 41 in Table I). Substitution of Gal{alpha}3 with a ß-GalNAc in 3-position (GalNAcß3-Gal{alpha}3[Fuc{alpha}2]Galß3GalNAcß4Galß4Glcß1Cer; Figure 2B, lane 4) abolished the binding, while the "ganglio-B" glycosphingolipid obtained by hydrolysis with jack bean ß-hexosaminidase (Gal{alpha}3[Fuc{alpha}2]Galß3GalNAcß4Galß4- Glcß1Cer; Figure 2B, lane 5) was recognized by the lectin. Furthermore, the MOA did not bind to the x2 glycosphingolipid (GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer; Figure 3B, lane 6) or to GlcNAcß3Galß4GlcNAcß3Galß4- Glcß1Cer or GalNAc{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer.



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Fig. 2. Binding of 125I-labeled MOA and EEA to pure glycosphingolipids on thin-layer chromatograms. Chemical detection by anisaldehyde (A) and autoradiograms obtained by binding of 125I-labeled MOA (B) and EEA (C). The glycosphingolipids were separated on aluminum-backed silica gel plates, using chloroform/methanol/water (60:35:8 v/v/v) as solvent system, and the binding assays were performed as described under Materials and methods. The lanes were as follows: lane 1, GalNAcß3-Gal{alpha}3Galß4Glcß1Cer (isoglobotetraosylceramide), 4 µg; lane 2, Gal{alpha}3(Fuc{alpha}2)Galß3(Fuc{alpha}4)GlcNAcß3Galß4Glcß1Cer (B7 type 1 heptaglycosylceramide), 4 µg; lane 3, GalNAc{alpha}3(Fuc{alpha}2)Galß4GlcNAc-ß3Galß4Glcß1Cer (A6 type 2 hexaglycosylceramide), 4 µg; lane 4, GalNAcß3Gal{alpha}3(Fuc{alpha}2)Galß3GalNAcß4Galß4Glcß1Cer, 4 µg; lane 5, Gal{alpha}3(Fuc{alpha}2)Galß3GalNAcß4Galß4Glcß1Cer, 4 µg. Autoradiography was for 6 h. The band marked x is a nonglycosphingolipid contaminant.

 


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Fig. 3. Binding of 125I-labeled MOA and EEA to pure glycosphingolipids on thin-layer chromatograms. Chemical detection by anisaldehyde (A) and autoradiograms obtained by binding of 125I-labeled MOA (B) and EEA (C). The glycosphingolipids were separated on aluminum-backed silica gel plates, using chloroform/methanol/water (60:35:8 v/v/v) as solvent system, and the binding assays were performed as described under Materials and methods. The lanes were as follows: lane 1, Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer, 4 µg; lane 2, Gal{alpha}3Gal{alpha}4Galß4Glcß1Cer, 4 µg; lane 3, Gal{alpha}3Galß4- GlcNAcß3Galß4Glcß1Cer (B5 pentaglycosylceramide), 4 µg; lane 4, Gal{alpha}3(Fuc{alpha}2)Galß3GlcNAcß3Galß4Glcß1Cer (B6 type 1 hexaglycosylceramide), 4 µg; lane 5, Gal{alpha}3Galß3GlcNAcß3Galß4- Glcß1Cer, 4 µg; lane 6, GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer (x2 pentaglycosylceramide), 4 µg. Autoradiography was for 12 h.

 


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Fig. 4. Binding of 125I-labeled EEA to pure glycosphingolipids on thin-layer chromatograms. Chemical detection by anisaldehyde (A) and autoradiogram obtained by binding of 125I-labeled EEA (B). The glycosphingolipids were separated on aluminum-backed silica gel plates, using chloroform/methanol/water (60:35:8 v/v/v) as solvent system, and the binding assays were performed as described under Materials and methods. The lanes were as follows: lane 1, GalNAc{alpha}3GalNAcß3Gal{alpha}4Galß4- Glcß1Cer (Forssman pentaglycosylceramide), 4 µg; lane 2, Fuc{alpha}2- Galß4GlcNAcß3Galß4Glcß1Cer (H5 type 2 pentaglycosylceramide), 4 µg; lane 3, Fuc{alpha}2Galß3(Fuc{alpha}4)GlcNAcß3Galß4Glcß1Cer (Leb-6 type 1 hexaglycosylceramide), 4 µg; lane 4, GalNAc{alpha}3(Fuc{alpha}2)Galß4GlcNAcß3- Galß4Glcß1Cer (A6 type 2 hexaglycosylceramide), 4 µg; lane 5, GalNAc{alpha}3(Fuc{alpha}2)Galß4(Fuc{alpha}3)GlcNAcß3Galß4Glcß1Cer (A7 type 2 heptaglycosylceramide), 4 µg; lane 6, Gal{alpha}3(Fuc{alpha}2)- Galß4GlcNAcß3Galß4Glcß1Cer (B6 type 2 hexaglycosylceramide), 4 µg; lane 7, Gal{alpha}3(Fuc{alpha}2)Galß3GlcNAcß3Galß4Glcß1Cer (B6 type 1 hexaglycosylceramide), 4 µg. Autoradiography was for 12 h.

 

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Table I. Binding of 125I-labeled Marasmius oreades lectin and Euonymus europaeus lectin to glycosphingolipids on thin-layer chromatograms

 
The EEA also bound to all glycosphingolipids with terminal Gal{alpha}3 (Nos. 16, 17, 24, 25, 28, 30, 40, and 41 in Table I), but not to the glycosphingolipid having a ß-GalNAc in 3-position of Gal{alpha}3 (GalNAcß3Gal{alpha}3(Fuc{alpha}2)Galß3- GalNAcß4Galß4Glcß1Cer; Figure 2B, lane 4). In contrast to MOA, the lectin of E. europaeus also interacted with glycosphingolipids with terminal {alpha}2-linked Fuc, as the H-5 type 2 glycosphingolipid (Fuc{alpha}2Galß4GlcNAcß3Gal- ß4Glcß1Cer; Figure 4B, lane 2), the H-5 type 1 glycosphingolipid (Fuc{alpha}2Galß3GlcNAcß3Galß4Glcß1Cer), and fucosyl-gangliotetraosylceramide (Fuc{alpha}2Galß3GalNAcß4Galß4Glcß1Cer). However, no binding to glycosphingolipids with a Fuc {alpha}4-linked or {alpha}3-linked to the penultimate GlcNAc, as the Lea-5 glycosphingolipid (Galß3[Fuc{alpha}4]GlcNAcß3Galß4Glcß1Cer; Figure 5B, lane 1) or the Lex-5 glycosphingolipid (Galß4[Fuc{alpha}3]GlcNAcß3Galß4Glcß1Cer) was obtained. Moreover, the Leb-6 glycosphingolipid (Fuc{alpha}2Galß3[Fuc{alpha}4]GlcNAcß3Galß4Glcß1Cer; Figure 4B, lane 3) was also nonbinding despite the presence of a terminal {alpha}2-linked Fuc.



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Fig. 5. Binding of 125I-labeled EEA to pure glycosphingolipids on thin-layer chromatograms. Chemical detection by anisaldehyde (A) and autoradiogram obtained by binding of 125I-labeled EEA (B). The glycosphingolipids were separated on aluminum-backed silica gel plates, using chloroform/methanol/water (60:35:8 v/v/v) as solvent system, and the binding assays were performed as described under Materials and methods. The lanes were as follows: lane 1, Galß3(Fuc{alpha}4)GlcNAcß3Galß4Glcß1Cer (Lea-5 pentaglycosylceramide), 4 µg; lane 2, Gal{alpha}3Galß4GlcNAcß3 Galß4Glcß1Cer (B5 pentaglycosylceramide), 4 µg; lane 3, GalNAcß3Galß-4GlcNAcß3Galß4Glcß1Cer (x2 pentaglycosylceramide), 4 µg; lane 4, Gal{alpha}3Galß4GlcNAcß6 (NeuGc{alpha}3Galß4GlcNAcß3) Galß4GlcNAcß3- Galß4Glcß1Cer, 4 µg; lane 5, GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer, 4 µg. Autoradiography was for 8 h.

 
Binding to the x2 glycosphingolipid (GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer; Figures 3C, lane 6, and 5B, lane 3), and to GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer (Figure 5B, lane 5), was also obtained with the EEA, whereas GalNAc{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer was not recognized.

Other glycosphingolipids with terminal GalNAc{alpha}3 or with terminal Gal{alpha}4 or NeuAc{alpha}3 were not recognized by either lectin.

Relative binding affinities
To estimate the relative affinity of MOA for various binding-active glycosphingolipids, the binding of radiolabeled lectin to serial dilutions of glycosphingolipids in microtiter wells was determined. As shown in Figure 6 the lectin preferentially interacted with the B6 type 2 glycosphingolipid (Gal{alpha}3[Fuc{alpha}2]Galß4GlcNAcß3Galß4Glcß1Cer) with a half maximal binding at approximately 10 ng/well. The lectin also bound to the B5 glycosphingolipid (Gal{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer, half maximal binding at 200 ng/well), whereas the binding to the B6 type 1 (Gal{alpha}3- [Fuc{alpha}2]Galß3GlcNAcß3Galß4Glcß1Cer) and the A6 type 2 glycosphingolipid (GalNAc{alpha}3[Fuc{alpha}2]Galß4GlcNAcß3- Galß4Glcß1Cer) was negligible.



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Fig. 6. Binding of 125I-labeled MOA to serial dilutions of glycosphingolipids in microtiter wells. The assay was done as described in the Materials and methods section. Data are presented as mean values of triplicate determinations, after subtraction of background values.

 
When binding of the lectin from E. europaeus to the B6 type 2 glycosphingolipid, the B5 glycosphingolipid and the H5 type 2 glycosphingolipid in microtiter wells was attempted, only the B6 type 2 glycosphingolipid gave a signal. Therefore, binding assays using dilutions of glycosphingolipids on thin-layer plates followed by densitometry (Figure 7) was utilized to be able to compare the relative affinity of EEA for binding-active glycosphingolipids. The B6 type 2 glycosphingolipid was the preferred ligand for this lectin, followed by the B6 type 1 glycosphingolipid and the H5 type 2 glycosphingolipid, and the B5 glycosphingolipid gave almost no signal.




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Fig. 7. Binding of 125I-labeled EEA to dilutions of glycosphingolipids on thin-layer plates. (A) Autoradiograms obtained by binding of 125I-labeled EEA using the chromatogram binding assay, as described in Materials and methods. The lanes were dilutions (0.5–0.002 µg) of Gal{alpha}3(Fuc{alpha}2)- Galß4GlcNAcß3Galß4Glcß1Cer (B6 type 2 glycosphingolipid), Gal{alpha}3- Galß4GlcNAcß3Galß4Glcß1Cer (B5 glycosphingolipid), Gal{alpha}3- (Fuc{alpha}2)Galß3GlcNAcß3Galß4Glcß1Cer (B6 type 1 glycosphingolipid), and Fuc{alpha}2Galß4GlcNAcß3Galß4Glcß1Cer (H5 type 2 glycosphingolipid). The results from one representative experiment out of three is shown. (B) Quantification of binding by densitrometry. The autoradiogram in (A) was analyzed using the NIH Image program.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Considered together, the glycosphingolipid binding assays demonstrate that the MOA binds to Gal{alpha}3-terminated glycosphingolipids with a preference for type 2 chains. Further substitution of the terminal Gal{alpha}3 with a ß-GalNAc in 3-position is, however, not tolerated. The microtiter well assay demonstrates that the MOA has a preference for the B6 type 2 glycosphingolipid (Gal{alpha}3[Fuc{alpha}2]Galß4GlcNAc- ß3Galß4Glcß1Cer) over the B5 glycosphingolipid (Gal{alpha}3- Galß4GlcNAcß3Galß4Glcß1Cer), suggesting that the {alpha}2-linked Fuc is accommodated in the carbohydrate binding site and provides additional interactions.

The B6 type 2 glycosphingolipid is also the ligand of choice for the lectin from E. europaeus. However, this lectin has a broader binding specificity. The EEA has no strict preference for type 2 chains because it also binds avidly to the B6 type 1 glycosphingolipid (Gal{alpha}3[Fuc{alpha}2]Galß3Glc NAcß3Galß4Glcß1Cer). Furthermore, the H5 type 2 glycosphingolipid (Fuc{alpha}2Galß4GlcNAcß3Galß4Glcß1Cer), devoid of a terminal {alpha}3-linked Gal, is preferred over the B5 glycosphingolipid, demonstrating an even more significant contribution to the binding affinity by the {alpha}2-linked Fuc than for the MOA.

Examination of molecular models of the B6 type 2 and B6 type 1 glycosphingolipids provides a structural rationale behind the different observed affinities for both lectins. The major difference between the two structures lies in an approximately 180° rotation of the GlcNAcß3 residue, resulting in the acetamido moiety of this sugar in B6-2 being found in the position of the hydroxymethyl group in B6-1 and vice versa (Figure 8). Thus, the bulky and hydrophobic acetamido moiety is seen to interact significantly with the Fuc{alpha}2 residue in B6-1, whereas it is pointing away from the binding epitope in B6-2, suggesting that structural interference from this group is the major reason for the reduced lectin affinities for B6-1. Additionally, it is anticipated that binding epitope presentation effects should be a contributing factor because different Glcß1Cer linkage conformations for B6-1 and B6-2 would have to be adopted for the epitope to be presented optimally.



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Fig. 8. Molecular models of the terminal tetrasaccharides of the B6 type 1 glycosphingolipid (Gal{alpha}3[Fuc{alpha}2]Galß3GlcNAcß3Galß4Glcß1Cer) (left) and the B6 type 2 glycosphingolipid (Gal{alpha}3[Fuc{alpha}2]Galß4GlcNAcß3- Galß4Glcß1Cer) (right). The only significant differences between the structures reside in the approximately 180° rotation of the GlcNAcß3 residue.

 
No binding of the EEA to the Leb-6 glycosphingolipid (Fuc{alpha}2Galß3[Fuc{alpha}4]GlcNAcß3Galß4Glcß1Cer) was obtained, despite the presence of a terminal Fuc{alpha}2. This is most likely due to an inhibitory effect of the {alpha}Fuc in 4-position of the GlcNAc, which would cover the penultimate Galß3 residue on the less important right side of the epitope as viewed in Figure 8. The B7 type 1 glycosphingolipid (Gal{alpha}3[Fuc{alpha}2]Galß3[Fuc{alpha}4]GlcNAcß3Galß4Glcß1Cer), on the other hand, was recognized due to the compensatory effect caused by the addition of the more critical terminally located {alpha}3-linked Gal seen on the top left side. Even though the Leb-6 glycosphingolipid was not tested against the MOA the binding of the B7 type 1 glycosphingolipid indicates that this lectin behaves similarly to the EEA.

The more tolerant nature of the lectin from E. europaeus is also evidenced by the fact that it also binds to the x2 glycosphingolipid (GalNAcß3Galß4GlcNAcß3Galß4Glc- ß1Cer) and GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. However, no binding of this lectin to GalNAc{alpha}3Galß4 GlcNAcß3Galß4Glcß1Cer was obtained.

Although both MOA and EEA readily bound to Gal{alpha}3Gal{alpha}3Galß4Glcß1Cer, binding to the iso-structure Gal{alpha}3Gal{alpha}4Galß4Glcß1Cer was only observed when using freshly labeled lectins. A reduced accessibility of the terminal Gal{alpha}3 of the latter glycosphingolipid is likely, because molecular modeling of other Gal{alpha}4Gal-containing glycosphingolipids has demonstrated that the Gal{alpha}4Gal sequence imposes a bend in the carbohydrate chain (Strömberg et al., 1991Go).

In a previous study we demonstrated that toxin A of C. difficile, human natural anti {alpha}-galactosyl IgG, and the monoclonal antibody Gal-13 all recognized Gal{alpha}3Galß4-GlcNAcß-terminated glycosphingolipids and also bound to three glycosphingolipids with the different terminal substituents and anomerity, that is, GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer (x2 glycosphingolipid), GalNAc{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer, and GlcNAcß3Galß4-GlcNAcß3Galß4Glcß1Cer (Teneberg et al., 1996Go). The basis for this surprising cross-binding was explained by examination of minimum energy conformations, demonstrating that the terminal parts of GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer, GalNAc{alpha}3Galß4GlcNAcß3Galß4- Glcß1Cer, and GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer may adopt a spatial topography on one side almost identical to that of the terminal part of Gal{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer.

Comparison of the binding preferences of the MOA and the EEA with the binding of toxin A of C. difficile and the Gal-13 monoclonal antibody shows several differences. Although all proteins bind to Gal{alpha}3Galß4GlcNAcß3- Galß4Glcß1Cer (B5 glycosphingolipid), the two lectins prefer glycosphingolipids with terminal blood group B determinants, whereas toxin A of C. difficile and the Gal-13 monoclonal antibody do not tolerate substitution with an {alpha}Fuc in 2-position of the penultimate Gal. Furthermore, terminal Gal{alpha}3 on a type 1 chain (Gal{alpha}3Galß3GlcNAcß3- Galß4Glcß1Cer) is also recognized by the lectins but not by the toxin or antibody. On the other hand, toxin A and the Gal-13 antibody interacts with GalNAc{alpha}3Galß4GlcNAc- ß3Galß4Glcß1Cer. This glycosphingolipid is not bound by the two lectins, although EEA binds to GalNAcß3Galß4- GlcNAcß3Galß4Glcß1Cer (x2 glycosphingolipid) and GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer.

These differences between the two lectins, on one hand, and toxin A and the Gal-13 antibody, on the other, may be rationalized as follows. The Gal(NAc){alpha}3Gal glycosidic linkage is conformationally rather restricted, allowing essentially only the conformation seen in Figure 8 (Imberty et al., 1995Go), whereas the Glc(Gal)NAcß3Gal linkage is more flexible (Imberty et al., 1991Go), allowing several energetically favorable conformations. These facts indicate that a free 2-OH of the terminal Gal{alpha}3 is essential for both lectins when the proper conformation of the Gal{alpha}3Gal glycosidic linkage is assumed and that the EEA but not the MOA accepts a reoriented Glc(Gal)NAcß3 to accommodate the bulky acetamido moiety of these residues. In line with this reasoning it should also be noted that although the B6 type 2 glycoshingolipid was the preferred ligand for both lectins, none of them bound to the A6 type 2 glycosphingolipid (GalNAc{alpha}3[Fuc{alpha}2]Galß4GlcNAcß3Galß4Glcß1Cer). However, both toxin A and the Gal-13 antibody lack the strict requirement for a free 2-OH on the terminal sugar residue, thus explaining the observed differences in binding even though the same conformational restrictions apply (Teneberg et al., 1996Go).

Further elucidation of the interactions involved in the carbohydrate binding by MOA and EEA may be obtained by X-ray crystallography. Efforts to crystallize the lectins of M. oreades and E. europaeus, alone and in complex with Gal{alpha}3Galß4GlcNAc saccharides, are currently under way.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Reference glycosphingolipids
The glycosphingolipids used in the binding assays were isolated at the Institute of Medical Biochemistry, Göteborg, following the protocol described by Karlsson (1987)Go. In brief, the tissues were lyophilized, followed by extraction in two steps with chloroform and methanol (2:1 and 1:9, v/v) in a Soxhlet apparatus. The material obtained was pooled and subjected to mild alkaline hydrolysis, followed by dialysis and purification on a silicic acid column. Thereafter, acid and nonacid glycolipids were separated on a DEAE-cellulose column. The nonacid fraction was acetylated and separated from alkali-stable phospholipids on a second silicic acid column. After deacetylation, final purification of the nonacid glycosphingolipids was done on DEAE- cellulose and silicic acid columns. The pure glycosphingolipids were in general obtained by acetylation of the total glycosphingolipid fractions, separation by repeated chromatographies on silicic acid columns, and by high-performance liquid chromatography. The pure compounds were characterized by mass spectrometry, proton nuclear magnetic resonance, and degradation studies as outlined in Teneberg et al. (1996)Go. Glycosphingolipid no. 11 in Table I was a kind gift of Dr. G.C. Hansson, Göteborg University, Sweden. Glycosphingolipids nos. 17 and 18 were produced from nos. 25 and 26, respectively, by incubation in 0.05 M HCl at 80°C for 2 h. Glycosphingolipid no. 20 was prepared from sialyl-neolactohexaosylceramide of rabbit thymus by mild acid hydrolysis (1% acetic acid at 100°C for 1 h), followed by hydrolysis with ß-galactosidase from Streptococcus pneumoniae (Oxford Glycosystems, Abingdon, UK) according to the protocol of the manufacturer. Glycosphingolipid no. 30 was produced from no. 31 by hydrolysis with jack bean ß-hexosaminidase (Glyko, Novato, CA), according to the manufacturer's instructions. Sialyl-Lex hexaglycosylceramide (no. 38) was purchased from ARC (Edmonton, Canada).

Lectins
EEA was purchased from Vector Laboratories (Burlingame, CA). The lectin from M. oreades was isolated by the original method described by Winter et al. (2002)Go.

Labeling
The lectins were diluted to 1 mg/ml in phosphate buffered saline, pH 7.3 (PBS). Aliquots of 100 µg were labeled with 125I, using Na125I (100 µCi/ml; Amersham Pharmacia Biotech, Little Chalfont, UK), according to the IODO-GEN protocol of the manufacturer (Pierce, Rockford, IL), giving approximately 5x103 cpm/µg protein.

Thin-layer chromatography
Thin-layer chromatography was performed on glass- or aluminum-backed silica gel 60 high-performance thin-layer chromatography plates (Merck, Darmstadt, Germany), using chloroform/methanol/water (60:35:8, v/v/v) as solvent system. Chemical detection was done with anisaldehyde (Waldi, 1962Go).

Chromatogram binding assay
Binding of radiolabeled lectin to glycosphingolipids separated on thin-layer plates was done as previously described (Teneberg et al., 1994Go). Mixtures of glycosphingolipids (20–40 µg/lane) or pure compounds (0.002–4 µg/lane) were separated on aluminum-backed silica gel plates. Thereafter, the chromatograms were treated with 0.5% (w/v) polyisobutylmethacrylate (Aldrich Chemical, Milwaukee, WI) in diethylether for 1 min. After drying, the chromatograms were soaked in PBS containing 2% bovine serum albumin (w/v), 0.1% NaN3 (w/v), and 0.1% Tween 20 (w/v) (solution I) for 2 h at room temperature. Thereafter, suspensions of 125I-labeled lectins (approximately 2x103 cpm/µl) diluted in solution I were gently sprinkled over the plates and incubated for 2 h at room temperature, followed by washing six times with PBS.

Autoradiography was performed for 12–24 h using XAR-5 X-ray films (Eastman Kodak, Rochester, NY) with an intensifying screen. For densitometry, selected autoradiograms were replicated using a CCD camera (Dage-MTI, Michigan City, IN), and analysis of the images was performed using the public domain NIH Image program (developed at the U.S. National Institutes of Health; available online at http://rsb.info.nih.gov/nih-image).

Microtiter well binding assay
The microtiter well binding assay was performed as previously described (Teneberg et al., 1994Go). In short, serial dilutions (each dilution in triplicate) of pure glycosphingolipids in methanol were applied in microtiter wells (Falcon 3911; Becton Dickinson Labware, Oxnard, CA). When the solvent had evaporated, the wells were blocked for 2 h with 200 µl solution I. Thereafter, 50 µl of radiolabeled lectin diluted in solution I (approximately 2x103 cpm/µl) were added per well and incubated over night at room temperature. After washing six times with PBS, the wells were cut out and the radioactivity counted in a gamma counter.

Molecular modeling
Minimum energy conformers for several of the glycosphingolipids listed in Table I were produced within the Quanta2000/CHARMm22 modeling package from Accelrys. Glycosidic dihedral angles for minimum energy conformers of constituent di- or trisaccharides were taken from the literature (Imberty et al., 1991Go, 1995Go; Teneberg et al., 1996Go).


    Acknowledgements
 
This study was supported by the Wallenberg Foundation, the National Institutes of Health (I.J.G), the Swedish Medical Research Council (Grant No.12628 to S.T.), the Swedish Cancer Foundation (S.T.), and the Lundberg Foundation.

The glycosphingolipid nomenclature follow the recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN for lipids: Eur. J. Biochem. [1977] 79, 11–21; J. Biol. Chem. [1982] 257, 3347–3351; and J. Biol. Chem. [1987] 262, 13–18). It is assumed that Gal, Glc, GlcNAc, GalNAc, NeuAc, and NeuGc are of the D-configuration; Fuc of the L-configuration; and all sugars present in the pyranose form.

1 To whom correspondence should be addressed; e-mail: susann.teneberg{at}medkem.gu.se Back


    Abbreviations
 
EEA, Euonymus europaeus lectin; MOA, Marasmius oreades lectin; PBS, phosphate buffered saline.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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