Structural characterization of the pectic polysaccharide rhamnogalacturonan II: evidence for the backbone location of the aceric acid-containing oligoglycosyl side chain

Monomeric rhamnogalacturonan II (mRG-II) was isolated from red wine and the reducing-end galacturonic acid of the backbone converted to L-galactonic a cid by treatment with NaBH4. The resulting product (mRG-II'ol) was treated with a cell-free extract from Penicillium daleae, a fungus that has been sh own to produce RG-II-fragmenting glycanases. The enzymatically generated pr oducts were fractionated by size-exclusion and anion-exchange chromatograph ies and the quantitatively major oligosaccharide fraction isolated. This fr action contained structurally related oligosaccharides that differed only i n the presence or absence of a single Kdo residue. The Kdo residue was remo ved by acid hydrolysis and the resulting oligosaccharide then characterized by 1- and 2D H-1 NMR spectroscopy, ESMS, and by glycosyl-residue and glyco syl-linkage composition analyses. The results of these analyses provide evi dence for the presence of at least two structurally related oligosaccharide s in the ratio similar to 6:1. The backbone of these oligosaccharides is co mposed of five (1 --> 4)-linked alpha-D-GalpA residues and a (1 --> 3)-link ed L-galactonate. The (1 --> 4)-linked GalpA residue adjacent to the termin al non-reducing GalpA residue of the backbone is substituted at O-2 with an apiosyl-containing side chain. beta-L-Araf-(1 --> 5)-beta-D-DhapA is likel y to be linked to O-3 of the GalpA residue at the non-reducing end of the b ackbone in the quantitatively major oligosaccharide and to O-3 of a (1 --> 4)-linked GalpA residue in the backbone of the minor oligosaccharide. Furth ermore, the results of our studies have shown that the enzymically generate d aceryl acid-containing oligosaccharide contains an alpha-linked aceryl ac id residue and a beta-linked galactosyl residue. Thus, the anomeric linkage s of these residues in RG-II should be revised. (C) 2000 Elsevier Science L td. All rights reserved.