3 Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606; and 4 Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0606
Received on July 31, 2003; revised on August 26, 2003; accepted on August 27, 2003
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Abstract |
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Key words:
agglutinin
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Gal1,3Galß1,4GlcNAc
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Marasmius oreades
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mushroom lectin
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serum glycoproteins
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Introduction |
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We have recently elucidated the carbohydrate binding specificity of a blood group Bspecific lectin from the mushroom Marasmius oreades (MOA) (Winter et al., 2002). Binding studies by the techniques of hapten inhibition of agglutination and precipitation, and hapten binding in solution by isothermal titration calorimetry established that MOA possesses an extended binding site for the Gal
1, 3Galß1,4GlcNAc structure, in contrast to other B-specific lectins, such as the Griffonia simplicifolia B4 isolectin (Goldstein and Winter, 1999
), which recognize only the
-galactosyl end group. Carbohydrate binding studies also revealed a high affinity of MOA for the type B branched trisaccharide (Gal
1,3[Fuc
1,2]Gal) and for the
1,3- but not the regioisomeric
1,2-,
1,4-, or
1,6-galactobioses.
Lectin affinity chromatography has served as a valuable technique for the separation and purification of glycans and glycoconjugates (Goldstein et al., 1997). This article describes the application of immobilized MOA for the fractionation of glycoconjugates from human sera and affords further insight into the carbohydrate specificity of this unique lectin.
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Results and discussion |
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These results indicate the presence of fucosylated blood group B structures on the fraction of 2-macroglobulin binding to immobilized MOA. Although UEA-I reacts weakly with H-1 structures (Fuc
1,2Galß1,3GlcNAc) (Petryniak and Goldstein, 1986
; Mollicone et al., 1996
), it cannot precipitate H-1 containing glycoconjugates, indicating that the majority of H glycans remaining after
-galactosidase digestion are undoubtedly of type 2 structure (Fuc
1,2Galß1,4GlcNAc), which interact strongly with this lectin. LTA also reacts with H-2 structures, but not at all with H-1 (Pereira and Kabat, 1974
), so its lack of reactivity in this case may reflect an insufficient quantity and/or improper presentation of H-2 structures. Indeed, Yan et al. (1997)
showed that certain glycoconjugates containing H-2 ligands reacted very poorly with immobilized LTA. Interestingly, sample B-2, after
-galactosidase digestion, failed to react with either fucose-binding lectin, suggesting that there may be individual differences in the quantity and/or structure of the
Gal determinants on the serum proteins, such as a preponderance of H-1 structures.
The presence of covalently linked ABO(H) blood group antigens on human plasma 2-macroglobulin and von Willebrand factor was reported by Matsui et al. (1993)
, using antibodies to the blood group substances. We have now confirmed this finding for
2-macroglobulin by isolation and partial characterization of this protein containing blood group B epitopes from the serum of type B individuals.
In some of the MOA-Sepharose-bound serum samples, relatively weak bands in the 5060 kDa range were identified by N-terminal amino acid sequence and by antibody staining of western blots as IgG heavy chains and antithrombin III. These bands, however, were present in type A and O serum also and did not stain on blots with biotinylated MOA. Furthermore, controls of fractions of serum bound to and eluted by diaminopropane from underivatized Sepharose also contained these two proteins (data not shown), indicating that a fraction of IgG and antithrombin III may exhibit affinity for the Sepharose matrix itself. Thus when using Sepharose-immobilized lectins to isolate lectin-bound components from serum or other biological fluids, a pretreatment of the serum with underivatized Sepharose is indicated.
The present article clearly demonstrates the utility of immobilized MOA for the affinity purification of glycoconjugates containing the Gal1,3Galß1,4GlcNAc trisaccharide sequence and serum components from human and animal sera. Immobilized MOA, with a unique and extended specificity for the Gal
1,3Galß1,4GlcNAc trisaccharide, should prove to be a very valuable probe for the detection, separation, and characterization of these biomedically important glycoconjugates.
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Materials and methods |
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Immobilization of MOA on Sepharose 4B
Cyanogen bromideactivated Sepharose 4B (1.5 g) was allowed to swell in 100 ml of 1 mM HCl solution for 30 min. The supernatant was removed, and the swollen beads (6 ml) were filtered on a sintered glass funnel, washed several times with 100 ml of 1 mM HCl and finally with 20 ml of 0.1 M sodium bicarbonate buffer, pH 8.3, containing 0.5 M sodium chloride. The beads were quickly transferred to a plastic bottle containing 16.2 mg MOA in the same buffer (10 ml) and 0.2 M lactose. The plastic bottle was shaken at room temperature for 6 h and at 4°C overnight. The beads were filtered on a sintered glass funnel, washed with the coupling buffer, and then shaken in 1 M ethanolamine (12 ml) at room temperature for 2.5 h to cap all of the unreacted iminocarbonate groups. The beads were filtered, washed alternately with the coupling buffer then sodium acetate buffer (0.1 M, pH 4.0, containing 0.5 M NaCl) four times. The beads contained 2.3 mg MOA per ml Sepharose 4B, based on MOA remaining in solution. A column (1 x 7 cm) packed with the beads was washed with 20 mM 1,3-diaminopropane to remove any noncovalently bound lectin, and finally washed with PBS containing 0.04% sodium azide.
Binding of various glycans, glycoconjugates, and serum samples to MOA-Sepharose: general procedure
The MOA-Sepharose column was loaded with glycan, glycoconjugate, or serum sample (0.65 ml) and washed with PBS until 280 nm absorbance was <0.01 (15 ml). The bound components were eluted with 20 mM 1,3-diaminopropane in 0.15 M NaCl (15 ml). The fractions collected (1 ml) were neutralized immediately with 1 M phosphoric acid (16 µl) and analyzed either by absorbance at 280 nm for protein or by phenol-sulfuric acid assay (Dubois et al., 1956
) for glycan. The column was finally washed thoroughly with PBS. The fractions containing the bound components were pooled and concentrated by Amicon ultrafiltration (Millipore, Bedford, MA) using a membrane (YM 10) of 10,000 molecular weight cutoff.
Digestion of proteins or serum fractions with green coffee bean -galactosidase or N-glycosidase F
For N-glycosidase F digestion, 20 µl of a solution of the MOA-bound serum fraction (at about 1 mg protein/ml) was heated 10 min at 80°C in 1% SDS. After cooling, 4 µl of 0.5 M phosphate buffer, pH 8.7, containing 2.5% Triton X-100 was added, followed by 1 µl N-glycosidase F from Chryseobacterium meningosepticum (5000 U/ml, Calbiochem, San Diego, CA). After overnight incubation at room temperature, 40 µl of SDSPAGE sample buffer was added, followed by heating in boiling water 5 min for SDSPAGE. Control samples were treated similarly, except for omission of the enzyme. For digestion with green coffee bean -galactosidase, the enzyme, 1 µl containing 50 IU/ml in ammonium sulfate suspension (Sigma G-8507) was added to 20 µl of MOA-bound serum fractions adjusted to pH 6.5 with 4 µl of 1.5 N citrate buffer and incubated at 37°C. The reaction was allowed to proceed for at least 24 h, and immediately denatured for gel electrophoresis. A solution of bovine thyroglobulin (2 mg) in pH 6.5 phosphate buffer was digested for 8 h with 18 µl (1 IU), followed by storage at 4°C.
Electrophoresis and western blotting
SDSPAGE analysis was carried out in 7.5% gels (0.8% cross-linked) using Tris/glycine buffer, pH 8.8, by the procedure of Laemmli (1970). The samples were denatured by boiling 5 min in buffer containing 1% SDS and 1% 2-mercaptoethanol. For western blotting, gels were equilibrated in 10 mM sodium carbonate buffer, pH 10, containing 20% methanol, electroblotted overnight at 10 V in the same buffer onto polyvinylidenedifluoride membranes (Immobilon-P, BioRad, Hercules, CA) using a BioRad Mini Trans-Blot cell. Blotted membranes were blocked with 5% nonfat dry milk and 0.5% Tween 20 in PBS for antibody probes or with 1% bovine serum albumin and 0.5% Tween 20 in PBS for lectin probes.
Ouchterlony double diffusion assays
Agar gel diffusion was carried out as described by Oudin (1980) in 50 x 9 mm snap lock petri dishes containing a layer of 0.85% agar in PBS
3 mm thick in which 6 mm diameter wells were cut, holding
50 µl of lectin or glycoprotein. Precipitin bands were observed visually with side lighting against a dark field.
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; e-mail: igoldste{at}umich.edu
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Abbreviations |
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References |
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