Isolation and structural characterization of endo-rhamnogalacturonase-generated fragments of the backbone of rhamnogalacturonan I

Abstract

A combination of commercially available preparations of Aspergillus niger β- d-galactosidase, endo-α- l-arabinanase, α- l-arabinosidase, and endo-β- d-galactanase has been used to generate oligoglycosyl fragments of the backbone of rhamnogalacturonan I (RG-I) that had been isolated from the walls of suspension-cultured sycamore cells. The backbone-cleaving enzyme, which is present in the β- d-galactosidase preparation, only fragments the RG-I backbone when many of the neutral oligoglycosyl side chains have been removed by the other exo- and endo-glycanases. The oligosaccharides released from the backbone were separated from the partially fragmented RG-I and then purified, as their oligoglycosyl aldonic acids, by HPAEC-PAD. Those backbone fragments with degrees of polymerization (dp's) between 2 and 11 were characterized using one- and two-dimensional 1H NMR spectroscopy, electrospray mass spectrometry, and glycosyl-residue and glycosyl-linkage composition analyses. Two series of oligoglycosyl fragments were identified. The quantitatively predominant series has the structure α- d- Gal pA-(1 → 2)-α- l-Rha p-[ → 4)-α- d-Gal pA-(1 → 2)-α- l-Rha p-(1 → ] n -4- d-Gal pA, and the quantitatively minor series has the structure α- l-Rha p-[ → 4)-α- d-Gal pA-(1 → 2)-α- l-Rha p-(1 → ] n -4- d-Gal pA ( n = 1–5). Thus, the enzyme preparations contain an α- l-rhamnosidase in addition to the endo-rhamnogalacturonase. The products of the endo-rhamnogalacturonase provide additional evidence that the backbone of RG-I is composed of the diglycosyl repeating unit: → 4)-α- d-Gal pA-(1 → 2)-α- l-Rha p-(1 →. The endo-rhamnogalac-???