Arabidopsis β1,2-xylosyltransferase: substrate specificity and participation in the plant-specific N-glycosylation pathway.
β1,2-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 β1,2-linked N-acetylglucosamine (GlcNAc) residue at the nonreducing terminus of the α1,3-branched mannose (Man) residue (GlcNAcβ1,2-Manα1,3-Man; GNM3B) for activity. However, AtXYLT showed decreased activity with substrates that contained α1,3-fucose at the chitobiose core-GlcNAc or a terminal GlcNAc at the α1,6-branched Man residue of GlcNAcβ1,2-Man (GlcNAcβ1,2-Manα1,6-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 β1,4-linked galactose (Gal) residue at the nonreducing terminus of GlcNAcβ1,2-Man. These results indicate that a β1,2-linked GlcNAc residue at the nonreducing terminus of an α1,3-branched Man residue is necessary for AtXYLT activity and that mammalian-type β1,4-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 α1,3-fucosyltransferase and mannosidase II in planta.
International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan.
Hiroyuki
H
Kajiura
Toru
T
Okamoto
Ryo
R
Misaki
Yoshiharu
Y
Matsuura
Kazuhito
K
Fujiyama
D000117:Acetylglucosamine / Q000737:chemistry; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002240:Carbohydrate Sequence; D005643:Fucose / Q000737:chemistry; D005690:Galactose / Q000737:chemistry; D006031:Glycosylation; D007313:Insecta / Q000166:cytology; D008358:Mannose / Q000737:chemistry; D010430:Pentosyltransferases / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry*; D013379:Substrate Specificity; D014994:Xylose / Q000737:chemistry
D029681:Arabidopsis Proteins; D011134:Polysaccharides; D005643:Fucose; D014994:Xylose; D010430:Pentosyltransferases; C408586:beta 1,2-xylosyltransferase; D008358:Mannose; D000117:Acetylglucosamine; D005690:Galactose