Structural characterization of endo-glycanase-generated oligoglycosyl side chains of rhamnogalacturonan I

Abstract

Rhamnogalacturonan I (RG-I) has been isolated from the walls of suspension-cultured sycamore cells ( Acer pseudoplatanus), and additional structural features of the polysaccharide were elucidated. Treatment of RG-I with a purified endo-(1 → 5)-α- l-arabinanase released a series of arabinose-containing oligosaccharides with degrees of polymerization (dp's) between 2 and 20. These oligosaccharides were shown, by glycosyl-linkage composition analysis, to contain terminal, 5-, and (3 → 5)-linked Ara f residues. These results provide evidence that a branched arabinan is attached to the backbone of RG-I. RG-I was freed of 95% of its arabinosyl residues by treating the polysaccharide with a combination of endo-(1 → 5)-α- l-arabinanase and α- l-arabinosidase. No galacturonic acid was released by these enzymes, which is evidence that the arabinosyl-containing portions of the side chains do not contain galactosyluronic acid residues. The galactose-containing portions of the side chains of RG-I were not fragmented by an endo-(1 → 4)-β- d-galactanase. However, approximately 85% of the galactose and small amounts of galacturonic acid were released by digestion of arabinose-depleted RG-I with a combination of endo- and exo-β- d-galactanases. The galacturonic acid may have been released by small amounts of an exo-α-galactosyluronidase contaminating the galactanases. Treatment of RG-I with this mixture of endo- and exo-glycanases resulted in a relatively size-homogeneous, almost side chain-free backbone composed of the O-acetylated diglycosyl repeating unit → 4)-α- d-Gal pA-(1 → 2)-α- l-Rha p. A combination of 1H NMR spectroscopy and periodate oxidation established that the backbone repeating unit contained a single O-acetyl substituent on C-2 or C-3 of each galactosyluronic acid residue.