131,2-Xylosyltransferase (XYLT) is a plant-specific glycosyltransferase that contributes to the biosynthesis of N-glycoproteins in plants. However, the specificity of XYLT for N-glycans has not yet been completely clarified. To gain insights into the function of XYLT in the plant N-glycosylation pathway, we examined the acceptor substrate specificity of recombinant Arabidopsis XYLT (AtXYLT) using 2-aminopyridine-labeled N-glycans as the substrates and confirmed the N-glycans of Arabidopsis xylt mutant. Recombinant AtXYLT expressed in insect cells required the beta 1,2-linked N-acetylglucosamine (GlcNAc) residue at the nonreducing terminus of the alpha 1,3-branched mannose (Man) residue (GlcNAc beta 1,2-Man alpha 1,3-Man; GNM3B) for activity. However, AtXYLT showed decreased activity with substrates that contained alpha 1,3-fucose at the chitobiose core-GlcNAc or a terminal GlcNAc at the alpha 1,6-branched Man residue of GlcNAc beta 1,2-Man (GlcNAc beta 1,2-Man alpha 1,6-Man; GNM3A), whose ratios were 10% and 50% of the optimal substrate, GNM3B, respectively. Moreover, AtXYLT did not show any activity in the transfer of the Xyl residue to N-glycans that contained a mammalian-type beta 1,4-linked galactose (Gal) residue at the nonreducing terminus of GlcNAc beta 1,2-Man. These results indicate that a beta 1,2-linked GlcNAc residue at the nonreducing terminus of an alpha 1,3-branched Man residue is necessary for AtXYLT activity and that mammalian-type beta 1,4-linked Gal residue(s) on the same branch completely inhibit(s) the activity. Furthermore, N-glycan analysis showed that approximately 30% of the N-glycans carry the Xyl residue in the wild type. These findings suggest that AtXYLT acts on protein-bound N-glycans prior to alpha 1,3-fucosyltransferase and mannosidase II in planta. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.