Clarification of the structural features of Rhamnogalacturonan-I type polysaccharide purified from radish leaves

Abstract

Determining the structure of REPsingle bondI, an immunostimulatory polysaccharide fraction from radish leaves, is an important health objective. Herein, we show that REP-I contains nine different monosaccharides, including GalA (22.2%), Gal (32.6%), Ara (27.5%), and Rha (10.2%) as main sugars. REP-I was also reacted with β-glucosyl Yariv reagent (29.8%), suggesting the presence of the arabino-β-3,6-galactan. Furthermore, methylated-product analysis revealed that REP-I contains 13 different glycosyl linkages, including 4-linked GalpA (21.0%), 2,4-linked Rhap (7.0%), 4-linked Galp (5.8%), 5-linked Araf (10.1%), and 3,6-linked Galp (7.9%), which are characteristic of RG-I. Microstructural information was obtained by sequential degradation using four linkage-specific glycosylases and β-elimination, with fragments analyzed on the basis of sugar composition, methylation, and MS/MS spectra. The results show that the immunostimulatory activity of REP-I is possibly due to the structure of RG-I, which is composed of a main chain with repeating [→2)-Rhap-(1 → 4)-GalpA-(1→] linkage units and three side-chains: a branched α(1 → 5)arabinan, a β(1 → 4)galactan, and arabino-β-3,6-galactan, which are branched at the C(O)4 position of each Rha residue in the REP-I main chain.