ENZYMATIC MIDCHAIN BRANCHING OF POLYLACTOSAMINE BACKBONES IS RESTRICTED IN A SITE-SPECIFIC MANNER IN ALPHA-1,3-FUCOSYLATED CHAINS

Branched polylactosamines on animal cell surfaces are believed to cont ribute to multivalent interactions in cell adhesion and cell signallin g. Their biosynthesis proceeds via linear precursors that become branc hed by beta 1,6-GlcNAc transferases (IGnT6, GlcNAc to Gal). Previous w ork has identified the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) and the hexasaccharide Gal beta 1-4GlcNAc beta 1- 3 Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (4) as accepters for a r at serum enzyme activity (cIGnT6), which transfers GlcNAc beta 1-6 uni ts to the midchain galactose residues; Thereby, 1 is converted to the branched pentasaccharide Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)G al beta 1-4GlcNAc and 4 to the doubly branched octasaccharide Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc [Leppanen, A., Salminen, H., Zhu, Y., Maa heimo, H., Helin, J., Costello, C. E., & Renkonen, O. (1997) Biochemis try 36, 7026-7036]. Here we report that neither the alpha 1,3-fucose-c ontaining derivatives of 1 [Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4(Fu c alpha 1-3)GlcNAc and Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal b eta 1-4GlcNAc] nor a similar derivative of 4 [Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4GlcNac] were accepters for the rat serum cIGnT6 activity. Hence, the enzyme's branc h-forming action was completely prevented at sites in the immediate ne ighborhood of the fucosylated loci of the polylactosamines. In Gal bet a 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc, the inhibition of the branch-forming reaction was restrict ed to the fucose-carrying LacNAc unit; at the middle LacNAc, the branc hing proceeded normally. However, in the isomeric Gal beta 1-4(Fuc alp ha 1-3)GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc, t he fucose residue prevented branching completely at the middle LacNAc and almost completely at the reducing end LacNAc. In summary, alpha 1, 3-fucose residues in polylactosamine chains inhibited the cIGnT6 react ion in a site-specific manner, at the fucosylated LacNAc unit itself a nd also at sites one and two LacNAc units upstream, but not at the Lac NAc 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 alpha 1,3-fucosyl units, that can be removed afterward without harming the branched backbones.