Isolation and properties of amaranthin, a lectin which recognizes the T-(or cryptic T) antigen.

Abstract

A lectin (Amaranthin) present in the seeds of Amaranthus caudatus has been isolated by affinity chromatography on Gal$\beta$1, 3GalNAc$\alpha$-$O$-R-Synsorb. The lectin occurs as a homodimer with subunit molecular weight 33-36 kDa. Antibodies against amaranthin did not detect lectin in stem or leaf extracts of the A. caudatus plant. Selected amaranthin peptides were isolated and partially sequenced. Amaranthin formed a precipitate with several asialoglycoproteins which possess $O$-linked saccharides. Hapten inhibition studies indicted that the T-disaccharide and its $\alpha$-linked glycosides (Gal$\beta$1, 3GalNAc$\alpha$-$O$-R) were the best inhibitors of lectin precipitation. $N$-Acetylgalactosamine, the only monosaccharide which inhibited precipitation, was 350-fold less effective than the T-disaccharide. The C$\sp\prime$-4 hydroxyl group of the galactosyl moiety, and the C-4 hydroxyl and C-2 acetamido groups of the GalNAc unit are the most important loci for interaction of the lectin with the T-disaccharide. The cryptic T-antigen NANA$\alpha$2, 3Gal$\beta$1, 3GalNAc$\alpha$-$O$ was as potent an inhibitor as Gal$\beta$1, 3GalNAc$\alpha$-$O$. Equilibrium dialysis with (6-$\sp3$H) Gal$\beta$1, 3GalNAc$\alpha$-$O$-CH$\sb3$ indicated that amaranthin has one binding site per subunit. Fluorescence enhancement titrations with naphthalenesulfonic acid derivatives revealed that this lectin also exhibits hydrophobic-binding properties similar to legume lectins. Hapten inhibition assays were also conducted for peanut agglutinin (PNA). A comparison between the carbohydrate-binding specificities of amaranthin and PNA is presented to explain the differences in reactivity found for these two T-antigen-specific lectins. Histochemical studies were carried out with biotinylated amaranthin and human colonic tissues. Amaranthin strained 46 $\pm$ 21% of the lower half of colonic crypts, compared with 7 $\pm$ 2% of the upper half (n = 23). The lectin bound to 97 $\pm$ 2% of 13 adenocarcinomas. Amaranthin bound (70 $\pm$ 21%) to normal-margin tissues from familial polyposis coli patients (n = 12) with greater frequency and in distinct locations within the crypts compared to true normals. These results suggest that amaranthin may be a potential diagnostic reagent for colon cancer and familial polyposis coli.