Structure and Biosynthesis of L-Fucosylated Arabinogalactan-Proteins in Cruciferous Plants

Abstract

Yamamoto (1982), surveying blood group ABH-active substances in vegetable foods, found potent blood group H-like activity inhibiting hemagglutination of human type O erythrocytes by eel anti-H sera in the polysaccharides from radish and turnip leaves. This finding prompted us to isolate and characterize L-fuc-osylated (L-Fuc) AGPs (F-AGPs) from leaves of radish, turnip, and rape (Nakamura et al 1984). The F-AGPs were purified from hot phosphate-buffered saline (PBS) extracts of these cruciferous plants by precipitation with three volumes of 99% ethanol and successive chromatography on DEAE-cellulose and on Sepharose 6B or Sephadex G-100. Elution with a linear gradient of NaHCO3 separated acidic AGPs from neutral and more acidic polysaccharides like starch and pectin. Treatment with α-amylase removed contaminating starch in the crude leaf polysaccharide fraction or unbound polysaccharide mixture. Rechromatography on anion exchange resin followed by gel filtration was effective for the preparation of higher purity AGPs differing in molecular masses. From radish, rape, and Sisybrium officinale leaves and radish primary roots, recoveries of total AGPs were 100–200 mg/kg fresh tissue weight. The purified AGPs were apparently homogenous upon ultracentrifugation and electrophoresis, and were subjected to serological and chemical analyses. Serological and immunological characterization of AGPs were conducted through precipitation reactions with eel anti-H sera, Aleuria aurantia L-Fuc-specific lectin (AAL), and rabbit antibodies raised against the β-1,6-galactotetraosyl group coupled to BSA, as well as with Yariv artificial antigen (Tsumuraya et al 1984a, Ogura et al 1985, Kikuchi et al 1993). Radish F-AGPs inhibited strong hemagglutination of human type O erythrocytes by eel anti-H sera and AAL, but their complete lack of such inhibitory activity on the hemagglutination by chicken anti-H sera and Ulex europeus lectin, as well as their antigenicity, clearly distinguished F-AGPs from human blood group O substances (Nakamura et al 1984). Application of these methods to the PBS extracts from growing organs of radish plants revealed the maximal expression of blood H-like activity in roots at the earlier stage (2–8 days after germination) of development, and its rapid disappearance toward maturation (Tsumuraya et al 1988). In a survey of blood group H-like activity in 14 species of cruciferous plants, leaf polysaccharides from seven species gave positive reactions, and Table 1 shows the distribution of blood group H-active F-AGPs in cruciferous plants. Leaf F-AGPs from four plants related to turnip were found to be positive in all the precipitation reactions tested. No blood group H-like activity could be detected in the PBS-extracts from leaves of cabbage and Brussels sprouts. Indeed, AGPs purified from a PBS-extract of cabbage leaves were totally devoid of L-Fuc. The F-AGPs were isolated from watercress leaves but failed to inhibit hemagglutination of human O erythrocytes by eel anti-H sera even at a concentration of 400 μg/ml, suggesting the possibility that L-Fuc residues were attached to α-L-Ara/ at a location different from the α-(1→2)-L-fucosidic linkage in the active polysaccharides.