Identification of a disaccharide side chain 2-O-alpha-D-galactopyranosyl-alpha-D-glucuronic acid in Arabidopsis xylan

Arabidopsis xylan consists of a linear chain of beta-1,4-linked D-xylosyl residues, about 10% of which are substituted with single residues of a-D-glucuronic acid (GlcA) or 4-O-methyl-alpha-D-glucuronic acid (MeGlcA) at O-2. In addition, about 60% of xylosyl residues are acetylated at O-2 and/or O-3. Previous studies have identified a number of genes responsible for elongation of the xylan backbone, addition of the GlcA substituents, and methylation of the GlcA residues. Yuan et al. (2013) have recently reported that the 2-O-and 3-O-monoacetylation of xylosyl residues in Arabidopsis xylan requires a DUF231 domain-containing protein, ESKIMO1 (ESK1), and proposed that ESK1 and its homologs are putative acetyltransferases responsible for xylan acetylation. It was noticed that the H-1 nuclear magnetic resonance (NMR) spectra of the acetylated xylan from the esk1 mutant and the wild-type Arabidopsis exhibited a prominent proton signal peak at 5.42 ppm in addition to resonances corresponding to known acetylated structural groups of xylan. Here, we performed detailed structural investigation of wild-type Arabidopsis acetylated xylan using 2-dimensional H-1-H-1 and H-1-C-13 NMR spectroscopy and found that the signal peak at 5.42 ppm in the 1H NMR spectrum was attributed to GlcA residues substituted at O-2 with a-D-galactose (Gal), indicating the presence of Gal-GlcA disaccharide side chains in Arabidopsis xylan. This finding was further supported by analysis of endoxylanase-digested xylan using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Our study demonstrates that Arabidopsis xylan contains Gal-GlcA disaccharide side chains in addition to GlcA, MeGlcA, and acetyl substitutions.