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
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Abstract |
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Key words:
Euonymus europaeus lectin
/
glycosphingolipid binding
/
Gal3Gal epitope
/
Marasmius oreades lectin
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Introduction |
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A recent communication described the isolation, characterization, and carbohydrate-binding properties of the lectin present in the mushroom Marasmius oreades (Winter et al., 2002). This lectin recognizes the Gal
3Galß4GlcNAc epitope but also binds to the branched blood group B epitope noted in the previous paragraph. The Gal
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
2Gal-) (Petryniak and Goldstein, 1986
, 1987
). 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
3Galß4GlcNAc-binding proteins studied previously.
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Results |
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Further binding assays were done using pure glycosphingolipids. The results are exemplified in Figures 24 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
3Galß4Glcß1Cer; Figure 2B, lane 1), the MOA bound to all glycosphingolipids with terminal Gal
3 (nos. 16, 17, 24, 25, 28, 30, 40, and 41 in Table I). Substitution of Gal
3 with a ß-GalNAc in 3-position (GalNAcß3-Gal
3[Fuc
2]Galß3GalNAcß4Galß4Glcß1Cer; Figure 2B, lane 4) abolished the binding, while the "ganglio-B" glycosphingolipid obtained by hydrolysis with jack bean ß-hexosaminidase (Gal
3[Fuc
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
3Galß4GlcNAcß3Galß4Glcß1Cer.
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Other glycosphingolipids with terminal GalNAc3 or with terminal Gal
4 or NeuAc
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 (Gal3[Fuc
2]Galß4GlcNAcß3Galß4Glcß1Cer) with a half maximal binding at approximately 10 ng/well. The lectin also bound to the B5 glycosphingolipid (Gal
3Galß4GlcNAcß3Galß4Glcß1Cer, half maximal binding at 200 ng/well), whereas the binding to the B6 type 1 (Gal
3- [Fuc
2]Galß3GlcNAcß3Galß4Glcß1Cer) and the A6 type 2 glycosphingolipid (GalNAc
3[Fuc
2]Galß4GlcNAcß3- Galß4Glcß1Cer) was negligible.
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Discussion |
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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 (Gal3[Fuc
2]Galß3Glc NAcß3Galß4Glcß1Cer). Furthermore, the H5 type 2 glycosphingolipid (Fuc
2Galß4GlcNAcß3Galß4Glcß1Cer), devoid of a terminal
3-linked Gal, is preferred over the B5 glycosphingolipid, demonstrating an even more significant contribution to the binding affinity by the
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 Fuc2 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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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 GalNAc3Galß4 GlcNAcß3Galß4Glcß1Cer was obtained.
Although both MOA and EEA readily bound to Gal3Gal
3Galß4Glcß1Cer, binding to the iso-structure Gal
3Gal
4Galß4Glcß1Cer was only observed when using freshly labeled lectins. A reduced accessibility of the terminal Gal
3 of the latter glycosphingolipid is likely, because molecular modeling of other Gal
4Gal-containing glycosphingolipids has demonstrated that the Gal
4Gal sequence imposes a bend in the carbohydrate chain (Strömberg et al., 1991
).
In a previous study we demonstrated that toxin A of C. difficile, human natural anti -galactosyl IgG, and the monoclonal antibody Gal-13 all recognized Gal
3Galß4-GlcNAcß-terminated glycosphingolipids and also bound to three glycosphingolipids with the different terminal substituents and anomerity, that is, GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer (x2 glycosphingolipid), GalNAc
3Galß4GlcNAcß3Galß4Glcß1Cer, and GlcNAcß3Galß4-GlcNAcß3Galß4Glcß1Cer (Teneberg et al., 1996
). The basis for this surprising cross-binding was explained by examination of minimum energy conformations, demonstrating that the terminal parts of GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer, GalNAc
3Galß4GlcNAcß3Galß4- Glcß1Cer, and GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer may adopt a spatial topography on one side almost identical to that of the terminal part of Gal
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 Gal3Galß4GlcNAcß3- 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
Fuc in 2-position of the penultimate Gal. Furthermore, terminal Gal
3 on a type 1 chain (Gal
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
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)3Gal glycosidic linkage is conformationally rather restricted, allowing essentially only the conformation seen in Figure 8 (Imberty et al., 1995
), whereas the Glc(Gal)NAcß3Gal linkage is more flexible (Imberty et al., 1991
), allowing several energetically favorable conformations. These facts indicate that a free 2-OH of the terminal Gal
3 is essential for both lectins when the proper conformation of the Gal
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
3[Fuc
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., 1996
).
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 Gal3Galß4GlcNAc saccharides, are currently under way.
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Materials and methods |
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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).
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, 1962).
Chromatogram binding assay
Binding of radiolabeled lectin to glycosphingolipids separated on thin-layer plates was done as previously described (Teneberg et al., 1994). Mixtures of glycosphingolipids (2040 µg/lane) or pure compounds (0.0024 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 1224 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., 1994). 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., 1991, 1995
; Teneberg et al., 1996
).
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Acknowledgements |
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The glycosphingolipid nomenclature follow the recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN for lipids: Eur. J. Biochem. [1977] 79, 1121; J. Biol. Chem. [1982] 257, 33473351; and J. Biol. Chem. [1987] 262, 1318). 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
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Abbreviations |
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References |
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