Photoaffinity labeling of the adenine binding sites of two Dolichos biflorus lectins

Abstract

Two differentially expressed lectins from the legume Dolichos biflorus, the seed lectin and a stem and leaf lectin (DB58), were photoaffinity-labeled at their adenine binding sites using the probe [2-3H]8-azidoadenine. Both heteromeric subunits I and II of the seed lectin and alpha and beta of DB58 were specifically labeled. This result, combined with the adenine binding site stoichiometries of two identical sites/seed lectin tetramer or one site/DB58 dimer, indicates that the adenine binding site resides at a heterologous subunit interface. Three radiolabeled peaks from seed lectin and one from DB58 were isolated from chymotryptic digests of the labeled lectins by reverse phase chromatography at pH 7.0. From these four peaks, six unique peptide sequences were determined. When aligned with the concanavalin A sequence, four of these peptides map to three loops in the metal binding domain of concanavalin A. The remaining two sequences represent carboxyl-terminal peptides unique to the D. biflorus lectins which may extend to the putative binding site from adjacent, heterologous subunits. It thus appears that the adenine binding sites of these D. biflorus lectins are within the metal binding domain and adjacent to the carbohydrate binding site.