RESUMO
Elucidation of protein-protein interactions (PPIs) is often very challenging and yields complex and unclear results. Lectin-glycoprotein interactions are especially difficult to study due to the noncovalent nature of the interactions and inherently low binding affinities of proteins to glycan ligands on glycoproteins. Here, we report a "ligand-directed labeling probe (LLP)"-based approach to fabricate protein probes for elucidating protein-glycoprotein interactions. LLP was designed with dual photoactivatable groups for the introduction of an alkyne handle proximal to the carbohydrate-binding pocket of lectins, Ricinus communis agglutinin 120 (RCA120) and recombinant human Siglec-2-Fc. In proof-of-principle studies, alkynylated lectins were conjugated with a photoreactive diazirine cross-linker and an environment-sensitive fluorophore, respectively, by the bioorthogonal click reaction. The modified RCA120 or Siglec-2-Fc was used for detecting the interaction with the target glycoprotein in the solution or endogenously expressed glycoproteins on live HeLa cells. We anticipate that the fabrication of these protein probes will accelerate the discovery of novel PPIs.
