MALDI-TOF MS evidence for the linking of flax bast fibre galactan to rhamnogalacturonan backbone

Fibre-specific (1 → 4)-β-galactan extracted from bast fibre peels of developing flax (Linum usitatissimum L.) stem has been studied to elucidate its structural details. The polysaccharide was characterized by NMR and subjected to partial degradation protocols, including chemical and enzymatic approaches. The oligosaccharide fragments obtained were fractionated by gel permeation chromatography and analyzed for their molecular mass with MALDI-TOF MS. The obtained data show that this flax galactan is a complex RG-I polysaccharide with variable side chain structures. The backbone is composed of the common GalA-Rha repeats with a high degree of branching. These side chains are mainly composed of β-1,4-linked Gal oligomers: (1) short branches of only one or two Gal residue(s); (2) long (linear) branches of up to 26 Gal residues; (3) mixed branches of between 3 and 12 Gal residues (possibly derived from longer linear side chains), that are resistant to galactanase cleavage; (4) side chains of at least 17 Gal residues, decorated with single Ara moieties. The linkage between RG backbone and galactan side chains was confirmed by the presence of fragments with (Rha-GalA)nHexm structure type. Neither chemical, nor enzymatic hydrolysis yielded oligomeric GalA residues, indicating that RG-I blocks are not interrupted by HGA regions. The polymer can be cleaved only partially by the rhamnogalacturonan hydrolase used, while the remaining part is resistant, probably due to peculiarities of side chain structure. Novel Rha-GalA oligomers were liberated by RG-hydrolase containing two or three Gal attached to Rha near the cleavage site. The native polymer is decorated by acetyl groups, with yet unknown distribution patterns. Treatment with purified and well-characterized galactanase does not change the hydrodynamic volume of flax galactan (despite considerable cleavage of Gal moieties), suggesting a complex “secondary” structure of the polymer.