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 alpha3-fucosylation of pure polylactosamines.
We report that isomeric monofucosylhexasaccharides, Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4(Fucalpha1-3) GlcNAc, Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4 GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 GlcNAc, and bifucosylhexasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 (Fucalpha1-3)GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4( Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4GlcNAc can be isolated in pure form from reaction mixtures of the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc with GDP-fucose and alpha1,3-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 [Galbeta1-4(Fucalpha1-3)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 GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1- 4Gl cNAc and Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4G lcN Ac and the tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc 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 beta1,6-GlcNAc transferase (Leppänen et al., Biochemistry, 36, 13729-13735, 1997).
Niemelä
R
R
Institute of Biotechnology, University of Helsinki and Department of Bioscience, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland.
Natunen
J
J
Penttilä
L
L
Salminen
H
H
Helin
J
J
Maaheimo
H
H
Costello
C E
CE
Renkonen
O
O
D000606:Amino Sugars / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002847:Chromatography, Agarose; D000939:Epitopes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D005647:Fucosyltransferases / Q000378:metabolism; D006031:Glycosylation; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D016256:Lewis X Antigen / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008895:Milk, Human / Q000201:enzymology; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D014909:Wheat Germ Agglutinins
D000606:Amino Sugars; D000939:Epitopes; D016256:Lewis X Antigen; D011134:Polysaccharides; D014909:Wheat Germ Agglutinins; C066929:polylactosamine; D006154:Guanosine Diphosphate Fucose; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase