Abstract
Lectin from the mushroom Polyporus squamosus (PSL) has a unique carbohydrate-binding specificity for sialylated glycoconjugates containing Neu5Acalpha2,6Galbeta1,4Glc/GlcNAc trisaccharide sequences of asparagine-linked glycoproteins. In the present study, we elucidate the molecular basis for its binding specificity as well as establish a consistent source of this useful lectin using a bacterial expression system. cDNA cloning revealed that PSL contains a ricin B chain-like (QXW)(3) domain at its N-terminus that is composed of three homologous subdomains (alpha, beta and gamma). A recombinant lectin was expressed in Escherichia coli as a fully active, soluble form. It agglutinated rabbit erythrocytes and showed the highest affinity for Neu5Acalpha2,6Galbeta1,4GlcNAc, but not for the sialyl alpha2,3-linked isomer. We also investigated the structure-function relationship of PSL. A monomeric C-terminal deletion mutant lacking 40% of the lectin's molecular mass retained sugar-binding activity, indicating that the carbohydrate-binding sites are situated in the N-terminal portion of the lectin, whereas the C-terminal portion probably functions in oligomerization and structural stabilization. Mutant constructs that have single amino acid substitutions in the putative sugar-binding sites, based on sequence alignment with the ricin B-chain, indicate that the beta and gamma subdomains are most probably sugar-binding sites. The recombinantly expressed lectin will be a valuable reagent for the detection of the Neu5Acalpha2,6Galbeta1,4GlcNAc sequence of asparagine-linked glycans.