Fucosyltransferases produce N-glycans containing core l-galactose

l-Galactose (l-Gal) containing N-glycans and cell wall polysaccharides have been detected in the l-Fuc deficient mur1 mutant of Arabidopsis thaliana. The l-Gal residue is thought to be transferred from GDP-l-Gal, which is a structurally related analog of GDP-l-Fuc, but in vitrol-galactosylation activity has never been detected. In this study, we carried out preparative scale GDP-l-Gal synthesis using recombinant A. thaliana GDP-mannose-3',5'-epimerase. We also demonstrated the l-galactosylation assay of mouse α1,6-fucosyltransferase (MmFUT8) and A. thaliana α1,3-fucosyltransferase (AtFucTA). Both fucosyltransferases showed l-galactosylation activity from GDP-l-Gal to asparagine-linked N-acetyl-β-d-glucosamine of asialo-agalacto-bi-antennary N-glycan instead of l-fucosylation. In addition, the apparent K_m values of MmFUT8 and AtFucTA suggest that l-Fuc was preferentially transferred to N-glycan compared with l-Gal by fucosyltransferases. Our results clearly demonstrate that MmFUT8 and AtFucTA transfer l-Gal residues from GDP-l-Gal and synthesize l-Gal containing N-glycan in vitro.