Associating structural characteristics to immunomodulating properties of carrot rhamnogalacturonan-I fractions

Carrot rhamnogalacturonan-I (cRG-I) is a polydisperse polysaccharide with molecular weights of 7-250 kDa. Using size exclusion chromatography cRG-I was fractionated and pooled in fractions (PF1-6). All fractions contained the same RG-I monosaccharides and similar glycosidic linkages although in varying relative amounts. The main differences were in rhamnose substitution, arabinan- and galactan side chain length and in levels of acetylation and methyl esterification. Atomic force microscopy showed either spheric or elongated structures for cRG-I and its derived fractions. To gain insight in the structure-function relationship of cRG-I, the immunomodulatory effect of the six fractions and their saponified derivatives was assessed in vitro. All fractions, except PF2, dose-dependently stimulated TNFα, IL-6, IL-1β, IL-8 and IL-10 production in peripheral blood mononuclear cells (PBMCs) of three healthy donors. Cytokine levels were largely influenced by the Mw and degree of esterification of the individual fractions. Notably, the highest Mw fraction (100 kDa) displayed the most potent activity, which was strongly reduced after the removal of ester residues by saponification. In contrast, the 75 kDa Mw population (PF2) proved inactive while its saponified counterpart exhibited substantial immunomodulatory activity. This confirmed the role of ester residues on the immune profile of RG-I subpopulations.