Isolation and characterization of linear polylactosamines containing one and two site-specifically positioned Lewis x determinants: WGA agarose chromatography in fractionation of mixtures generated by random, partial enzymatic alpha 3-fucosylation of pure polylactosamines

We report that isomeric monofucosylhexasaccharides, Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAGlc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc, Gal bet a 1-4GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4G lcNAc and Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc, and bifucosylhexasaccharides Gal beta 1-4GlcNAc bet a 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNA beta 1-3Gal beta 1-4(Fuc alpha 1-3)Gl cNAc, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal1 beta 1-4GlcNAc beta 1- 3Gal beta 1-4(Fuc alpha 1-3)GlcNAc and Gal beta 1-4(Fuc alpha 1-3)GlcNAc be ta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4GlcNAc can be i solated in pure form from reaction mixtures of the linear hexasaccharide Ga l beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc with GDP-fucose and al,3-fucosyltransferases of human milk. The pure isomers we re characterized in several ways; H-1-NMR spectroscopy, for instance, revea led distinct resonances associated with the Lewis x group [Gal beta 1-4(Fuc alpha 1-3)GlcNAc] located at the proximal, middle, and distal positions of the polylactosamine chain. Chromatography on immobilized wheat germ agglut inin was crucial in the separation process used; the isomers carrying the f ucose at the reducing end GlcNAc possessed particularly low affinities for the lectin, Isomeric monofucosyl derivatives of the pentasaccharides GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc and Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc and the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc were also obtained in pure form, impl ying that the methods used are widely applicable. The isomeric Lewis x glyc ans proved to be recognized in highly variable binding modes by polylactosa mine-metabolizing enzymes, e.g., the midchain beta 1,6-GlcNAc transferase ( Leppanen et al., Biochemistry, 36, 13729-13735, 1997).