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

Abstract

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; 1 H-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 Galα1-3Galβ1-4GlcNAc-β1-3Galβ1-4GlcNAc 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).

A novel β1,3‐N‐acetylglucosaminyltransferase (β3Gn‐T8), which synthesizes poly‐N‐acetyllactosamine, is dramatically upregulated in colon cancer

“…Total cellular RNA was isolated from tissues using an RNeasy Ò Mini kit (Qiagen, Hilden, Germany). Complementary DNAs were synthesized using oligo(dT) [12][13][14][15][16][17][18] primers and the Superscript First-Strand Synthesis system (Invitrogen). Standard curves for the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, were generated by serial dilution of pCR2.1 (Invitrogen) DNA containing the GAPDH gene.…”

Enzymatic in vitro synthesis of I-branches of mammalian polylactosamines: generation of scaffolds for multiple selectin-binding saccharide determinants

“…This is perhaps not very surprising, because in aqueous solutions the galactose and the fucose of the Lex determinant are tightly associatedin fact, stacked on top of each other [28] -and many proteins recognizing readily unsubstituted LacNAc units are unable to bind Lex. Pertinent examples include exo-i-galactosidases (reviewed in [29]), endo-i-galactosidases of Bacteroides fragilis and Escherichia freundii (reviewed in [1]), iGnT [30], h3-sialyltransferase [31], wheat germ agglutinin [32,33] and Datura stramonium agglutinin [34]. The cIGnT activity of rat serum was also unable to generate branches to the fucose-containing pentasaccharide Lexi1-3%LacNAc [27].…”

“…Further experiments have enabled us to synthesize several kinds of i-type polylactosamines that are site-specifically h3-fucosylated at the reducing end terminus. One method is based on use of affinity chromatography on immobilized wheat germ agglutinin that directly separates Ri1-3%LacNAci1-3%Lex from all other isomers carrying the single h3-bonded fucose in different positions along the i-chain [33]. Another method of synthesis is based on h3-fucosylation of GlcNAci1-3%LacNAc before further elongation and modification of the chain [36,37].…”

The Acceptor and Site Specificity of α3-Fucosyltransferase V

“…Corresponding analyses of type 1 and type 2 sialopolylactosamines were performed after enzymatic desialylation. Analysis of the Fuc-TV reaction products of Lex␤1-3ЈLN␤1-3ЈLN was performed by B. fragilis endo-␤-galactosidase digestion (45).…”

“…Both features are characteristic to the sequence Gal␤1-4(Fuc␣1-3)GlcNAc␤1-OR (45,64,65), and neither of them is expected to be a property of the glycan Gal␤1-4GlcNAc␤1-4(Fuc␣1-3)GlcNAc. Hence, we conclude that LN␤1-4GlcNAc probably reacted with Fuc-TV mainly at the LN unit, and the reactivity of this unit was not appreciably inhibited by the presence of the proximal GlcNAc.…”