Cloning, expression in Escherichia coli and characterization of the recombinant Neu5Acα2,6Galβ1,4GlcNAc-specific high-affinity lectin and its mutants from the mushroom Polyporus squamosus

摘要

Lectin from the mushroom Polyporus squamosus (PSL) has a unique carbohydrate-binding specificity for sialylated glycoconjugates containing Neu5Acα2,6Galβ1,4Glc/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 (α, β and γ). A recombinant lectin was expressed in Escherichia coli as a fully active, soluble form. It agglutinated rabbit erythrocytes and showed the highest affinity for Neu5Acα2,6Galβ1,4GlcNAc, but not for the sialyl α2,3-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 β and γ subdomains are most probably sugar-binding sites. The recombinantly expressed lectin will be a valuable reagent for the detection of the Neu5Acα2,6Galβ1,4GlcNAc sequence of asparagine-linked glycans.