A bi-specific lectin from the mushroom Boletopsis grisea and its application in glycoanalytical workflows

The BLL lectin from the edible Japanese Kurokawa mushroom (Boletopsis leucomelaena) was previously reported to bind to N-glycans harboring terminal N-acetylglucosamine (GlcNAc) and to induce apoptosis in a leukemia cell line. However, its gene has not been reported. In this study, we used a transcriptomics-based workflow to identify a full-length transcript of a BLL functional ortholog (termed BGL) from Boletopsis grisea, a close North American relative of B. leucomelaena. The deduced amino acid sequence of BGL was an obvious member of fungal fruit body lectin family (Pfam PF07367), a highly conserved group of mushroom lectins with a preference for binding O-glycans harboring the Thomsen-Friedenreich antigen (TF-antigen; Gal beta 1,3GalNAc-alpha-) and having two ligand binding sites. Functional characterization of recombinant BGL using glycan microarray analysis and surface plasmon resonance confirmed its ability to bind both the TF-antigen and beta -GlcNAc-terminated N-glycans. Structure-guided mutagenesis of BGL's two ligand binding clefts showed that one site is responsible for binding TF-antigen structures associated with O-glycans, whereas the second site specifically recognizes N-glycans with terminal beta -GlcNAc. Additionally, the two sites show no evidence of allosteric communication. Finally, mutant BGL proteins having single functional bindings site were used to enrich GlcNAc-capped N-glycans or mucin type O-glycopeptides from complex samples in glycomics and glycoproteomics analytical workflows.

Automated summary

In this study, a full-length transcript of a functional ortholog of BLL, named BGL, was identified from Boletopsis grisea using a transcriptomics-based workflow. The deduced amino acid sequence of BGL belongs to the fungal fruit body lectin family, which has a preference for binding O-glycans and two ligand binding sites. Functional characterization of recombinant BGL confirmed its ability to bind both the TF-antigen and beta -GlcNAc-terminated N-glycans. Structure-guided mutagenesis showed that one binding site is responsible for O-glycans while the other specifically recognizes N-glycans. Mutant BGL proteins were used in glycomics and glycoproteomics workflows for enriching specific glycans or glycopeptides.