Structural characterisation of xyloglucan secreted by suspension-cultured cells of Nicotiana plumbaginifolia

Linkage analysis of a xyloglucan from the extracellular medium of suspension cultures of Nicotiana plumbaginifolia showed mostly 4-Glcp and 4,6-Glcp, terminal Xylp and 2-Xylp, and terminal Araf, along with ∼ 10% (w/w) O-acetyl groups, equivalent to ∼ 0.28 mol acetyl per mol of glycosyl residue. Methylation with methyl trifluoromethanesulfonate under neutral conditions, followed by re-methylation with CD3I under basic conditions, and conversion into partially methylated alditol acetates showed that O-acetyl groups were primarily attached to C-6 of ∼ 44% of the 4-Glcp backbone not substituted with Xylp residues and to C-5 of ∼ 15% of the terminal Araf residues. These positions of the O-acetyl groups were confirmed by 1H-NMR. Oligosaccharides generated by digestion of native xyloglucan with endo-(1 → 4)-β-glucanase were separated by a combination of gel-filtration chromatography and anion-exchange HPLC, and analysed by glycosyl linkage analysis and by electrospray ionisation-mass spectrometry (ESI-MS). The major oligosaccharide subunits were Glc4Xyl2 and Glc5Xyl2, of which 50–60% are substituted with one terminal Araf residue attached to 0–2 of a Xylp residue, and a further 20–25% are substituted with two terminal Araf residues attached to 0–2 of the Xylp residues. ESI-MS showed that many of the oligosaccharide subunits carried one, two and, occasionally three O-acetyl groups.