Incorporation of Fluorescent Nonnatural Amino Acid into Sialic Acid-Binding Lectin for Fluorescence Detection of Ligand-Binding

Abstract

Abstract Lectins are important not only as biological functional molecules but as useful probes for glycan analysis. Here, we developed fluorescent-labeled lectins showing fluorescence changes upon ligand-binding. BODIPYFL-aminophenylalanine was incorporated at eight respective positions near the ligand-binding site of a sialic acid-binding lectin in response to UAG codon in a cell-free translation system. Fluorescence imaging of SDS-PAGE gel indicated the successful expression of the fluorescent-labeled lectins containing BODIPYFL-linked amino acid. Fluorescence spectral measurements revealed that the fluorescent-labeled lectins showed significant increases in the fluorescence intensity upon the binding of sialyllactose for all the incorporation positions. Particularly, the fluorescence intensity increased 3.7-fold for the labeled lectin at Thr38 position. The increase in the fluorescence intensity could occur because the fluorescence of BODIPYFL is quenched by amino acid residues located near the ligand-binding site in the absence of ligand, but the ligand-binding reduces the fluorescence quenching by removing the BODIPYFL moiety from the ligand-binding site. The W31F substitution decreased the fluorescence change of the labeled lectin, suggesting that the Trp31 residue may partially contribute to the ligand-dependent quenching. The present strategy will be utilized for producing various fluorescent-labeled lectin probes showing ligand-dependent fluorescence change.