Antigenic determinants of a plant proteoglycan, the Gladiolus style arabinogalactan-protein

Abstract

Antiserum was raised in rabbits to the arabinogalactan-protein of Gladiolus style mucilage. This macromolecule has been characterized and has a structure consistent with a 1 .fwdarw. 3-linked .beta.-galactan backbone, with side branches of 1 .fwdarw. 6-linked .beta.-galactosyl residues, some of which carry terminal .alpha.-L-arabinofuranoside residues [Gleeson and Clarke]. The specificity of the antiserum has been investigated by immunoprecipitation with [3H]arabinogalactan-protein. The 3H label was introduced into the arabinogalactan-protein by oxidation of the terminal galactose residues with galactose oxidase, followed by reduction with NaB3H4. The antigenic specificity of the antiserum was shown to be directed towards the carbohydrate component of the arabinogalactan-protein. D-Galactose and L-arabinose were the most effective hapten inhibitors of the antiserum; other monosaccharides.sbd.N-acetyl-D-galactosamine, D-galactono-1,4-lactone, D-glucose, D-mannose, L-rhamnose, L-fucose and D-xylose, were all poor inhibitors. The antiserum showed preference for .beta.-galactosides over .alpha.-galactosides. Of the haptens examined, the disaccharide 6-O-.beta.-D-galactopyranosyl-D-galactopyranose, was the most potent inhibitor. The antigenic features of the arabinogalactan-protein were investigated by examining the interaction of the antiserum with chemically- and enzymically-modified arabinogalactan-protein. Also, the cross-reactivity of structurally-related polysaccharides and glycoproteins with the specific antiserum was assessed by a hemagglutination assay using erythrocytes coupled with specific antiserum. The results indicate that the dominant antigenic determinants of the arabinogalactan-protein are probably the side branches of 1 .fwdarw. 6-linked .beta.-galactose residues bearing the terminal .alpha.-L-arabinose residues.