Synthesis of poly-N-acetyllactosamine in core 2 branched O-glycans

Abstract

Poly-N-acetyllactosamine is a unique carbohydrate composed of N- acetyllactosamine repeats and provides the backbone structure for additional modifications such as sialyl Le(x). Poly-N-acetyllactosamines in mucin-type O-glycans can be formed in core 2 branched oligosaccharides, which are synthesized by core 2 β-1,6-N-acetylglucosaminyltransferase. Using a β- 1,4-galactosyltransferase (β4Gal-TI) present in milk and the recently cloned β-1,3-N-acetylglucosaminyltransferase, the formation of poly-N- acetyllactosamine was found to be extremely inefficient starting from a core 2 branched oligosaccharide, GlcNAcβ1-→6-(Galβ1→3)GalNAcα→R. Since the majority of synthesized oligosaccharides contained N-acetylglucosamine at the nonreducing ends, galactosylation was judged to be inefficient, prompting us to test novel members of the β4Gal-T gene family for this synthesis. Using various synthetic acceptors and recombinant β4Gal-Ts, β4Gal-TIV was found to be most efficient in the addition of a single galactose residue to GlcNAcβ1→6(Galβ1→3)GalNAcα→R. Moreover, β4Gal-TIV, together with β- 1,3-N-acetylglucosaminyltransferase, was capable of synthesizing poly-N- acetyllactosamine in core 2 branched oligosaccharides. On the other hand, β4Gal-TI was found to be most efficient for poly-N-acetyllactosamine synthesis in N-glycans. In contrast to β4Gal-TI, the efficiency of β4Gal- TIV decreased dramatically as the acceptors contained more N- acetyllactosamine repeats, consistent with the fact that core 2 branched O- glycans contain fewer and shorter poly-N-acetyllactosamines than N-glycans in many cells. These results, as a whole, indicate that β4Gal-TIV is responsible for poly-N-acetyllactosamine synthesis in core 2 branched O- glycans.