Synthesis of poly-N-acetyllactosamine in core 2 branched O-glycans - The requirement of novel beta-1,4-galtactosyltransferase IV and beta-1,3-N-acetylglucosaminyltransferase

Poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllacto samine repeats and provides the backbone structure for additional modificat ions such as sialyl Le(x), Poly-N-acetyllactosamines in mucin-type O-glycan s can be formed in core 2 branched oligosaccharides, which are synthesized by core 2 beta-1,6-N-acetylglucosaminyltransferase. Using a beta-1,4-galactosyltransferase (beta 4Gal-TI) present in milk and t he recently cloned beta-1,3-N-acetylglucosaminyltransferase, the formation of poly-N-acetyllactosamine was found to be extremely inefficient starting from a core 2 branched oligosaccharide, GlcNAc beta 1-->6-(Gal beta 1-->3)G alNAc alpha-->R. Since the majority of synthesized oligosaccharides contain ed N-acetylglucosamine at the nonreducing ends, galactosylation was judged to be inefficient, prompting us to test novel members of the beta 4Gal-T ge ne family for this synthesis. Using various synthetic accepters and recombi nant beta 4Gal-Ts, beta 4Gal-TIV was found to be most efficient in the addi tion of a single galactose residue to GlcNAc beta 1-->6(Gal beta 1-->3)Gal- NAc alpha-->R. Moreover, beta 4Gal-TIV, together with beta-1,3-N-acetylgluc osaminyltransferase, was capable of synthesizing poly-N-acetyllactosamine i n core 2 branched oligosaccharides. On the other hand, beta 4Gal-TI was fou nd to be most efficient for poly-N-acetyllactosamine synthesis in N-glycans , In contrast to beta 4Gal-TI, the efficiency of beta 4Gal-TIV decreased dr amatically as the accepters contained more N-acetyllactosamine repeats, con sistent with the fact that core 2 branched O-glycans contain fewer and shor ter poly-N-acetyllactosamines than N-glycans in many cells. These results, as a whole, indicate that beta 4Gal-TIV is responsible for poly-N-acetyllac tosamine synthesis in core 2 branched O-glycans.