Antigenic epitope for polyclonal antibody against a complement activating pectin from the roots of Angelica acutiloba Kitagawa

Abstract

A polyclonal antibody (anti-AR-2IIb-IgG) was developed against a complement activating pectin (AR-2IIb) which was purified from the roots of Angelica acutiloba Kitagawa. Neutral oligosaccharide–alditols were released from the “ramified” region (rhamnogalacturonan core possessing side chains rich in neutral sugars, PG-1) by lithium-degradation. The mixture significantly inhibited the reactivity of anti-AR-2IIb-IgG to PG-1 or AR-2IIb. Gel filtration and HPLC analysis indicated that the mixture contained two kinds of long oligosaccharide–alditols in addition to several kinds of shorter oligosaccharide–alditols, and both the long oligosaccharide–alditols had significant inhibitory activity against the reactivity of the antibody to PG-1. However, the short oligosaccharide–alditols had no effect. Methylation analysis suggested that one of the long oligosaccharide–alditols consisted of a β-(1→3,6)-galactan-like structure. The inhibitory activity of PG-1 against the reactivity of the antibody to PG-1 decreased significantly on exo-α- l-arabinofuranosidase digestion followed with exo-β- d-(1→3)-galactanase digestion, but not by exo-α- l-arabinofuranosidase or exo-β- d-galactosidase digestion. Although β- d-(1→6)-galacto-di- to tetra-saccharides showed no inhibitory activity, a mixture of oligosaccharides liberated from PG-1 by exo-β- d-(1→3)- galactanase digestion, still showed inhibitory activity. The oligosaccharide consisting mainly of terminal, 3- and 4-linked and 3,6-branched Gal, terminal Ara f and 4-OMe-GlcA inhibited the reactivity of the antibody to PG-1.