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 α3-fucosylation of pure polylactosamines

• Published in: Glycobiology (Oxford) 1999-05, Vol.9 (5), p.517-526
• Main Authors: Niemelä, Ritva, Natunen, Jari, Penttilä, Leena, Salminen, Heidi, Helin, Jari, Maaheimo, Hannu, Costello, Catherine E., Renkonen, Ossi
• Format: Article
• Language: English
• Subjects: linear polylactosamines; separation; site-specific; wheat germ agglutinin; α3-fucosylation
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/9.5.517

We report that isomeric monofucosylhexasaccharides, Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAc, Galβ1–4GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4GlcNAc and Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc, and bifucosylhexasaccharides Galβ1–4GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAc, Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAc and Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4GlcNAc can be isolated in pure form from reaction mixtures of the linear hexasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc with GDP-fucose and α1,3-fucosyltransferases of human milk. The pure isomers were characterized in several ways; 1H-NMR spectroscopy, for instance, revealed distinct resonances associated with the Lewis x group [Galβ1–4(Fucα1–3)GlcNAc] located at the proximal, middle, and distal positions of the polylactosamine chain. Chromatography on immobilized wheat germ agglutinin was crucial in the separation process used; the isomers carrying the fucose at the reducing end GlcNAc possessed particularly low affinities for the lectin. Isomeric monofucosyl derivatives of the pentasaccharides GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc and Galα1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc and the tetrasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc were also obtained in pure form, implying that the methods used are widely applicable. The isomeric Lewis x glycans proved to be recognized in highly variable binding modes by polylactosamine-metabolizing enzymes, e.g., the midchain β1,6-GlcNAc transferase (Leppänen et al., Biochemistry, 36, 13729–13735, 1997).