Enzymatic Midchain Branching of Polylactosamine Backbones Is Restricted in a Site-Specific Manner in α1,3-Fucosylated Chains

Branched polylactosamines on animal cell surfaces are believed to contribute to multivalent interactions in cell adhesion and cell signalling. Their biosynthesis proceeds via linear precursors that become branched by β1,6-GlcNAc transferases (IGnT6, GlcNAc to Gal). Previous work has identified the tetrasaccharide Galβ1−4GlcNAcβ1−3Galβ1−4GlcNAc (1) and the hexasaccharide Galβ1−4GlcNAcβ1−3Galβ1−4GlcNAcβ1−3Galβ1−4GlcNAc (4) as acceptors for a rat serum enzyme activity (cIGnT6), which transfers GlcNAcβ1−6 units to the midchain galactose residues. Thereby, 1 is converted to the branched pentasaccharide Galβ1−4GlcNAcβ1−3(GlcNAcβ1−6)Galβ1−4GlcNAc and 4 to the doubly branched octasaccharide Galβ1−4GlcNAcβ1−3(GlcNAcβ1−6)Galβ1−4GlcNAcβ1−3(GlcNAcβ1−6)Galβ1−4GlcNAc [Leppänen, A., Salminen, H., Zhu, Y., Maaheimo, H., Helin, J., Costello, C. E., & Renkonen, O. (1997) Biochemistry 36, 7026−7036]. Here we report that neither the α1,3-fucose-containing derivatives of 1 [Galβ1−4GlcNAcβ1−3Galβ1−4(Fucα1−3)GlcNAc and Galβ1−4(Fucα1−3)GlcNAcβ1−3Galβ1−4GlcNAc] nor a similar derivative of 4 [Galβ1−4GlcNAcβ1−3Galβ1−4(Fucα1−3)GlcNAcβ1−3Galβ1−4GlcNAc] were acceptors for the rat serum cIGnT6 activity. Hence, the enzyme's branch-forming action was completely prevented at sites in the immediate neighborhood of the fucosylated loci of the polylactosamines. In Galβ1−4GlcNAcβ1−3Galβ1−4GlcNAcβ1−3Galβ1−4(Fucα1−3)GlcNAc, the inhibition of the branch-forming reaction was restricted to the fucose-carrying LacNAc unit; at the middle LacNAc, the branching proceeded normally. However, in the isomeric Galβ1−4(Fucα1−3)GlcNAcβ1−3Galβ1−4GlcNAcβ1−3Galβ1−4GlcNAc, the fucose residue prevented branching completely at the middle LacNAc and almost completely at the reducing end LacNAc. In summary, α1,3-fucose residues in polylactosamine chains inhibited the cIGnT6 reaction in a site-specific manner, at the fucosylated LacNAc unit itself and also at sites one and two LacNAc units upstream, but not at the LacNAc units downstream from the fucosylated locus. These data imply that site-directed branching in polylactosamines is possible in vitro with the aid of specifically positioned α1,3-fucosyl units, that can be removed afterward without harming the branched backbones.