Development of a Calreticulin-targeting Glycan Ligand Based on a Hybrid Binding Concept.

Calreticulin (CRT), a chaperone that possesses both lectin and chaperone domains, is localized in the endoplasmic reticulum (ER). CRT has diverse functions and localizations; thus, CRT is a multifunctional protein. Particularly in the ER, CRT mainly aids in the proper folding of nascent glycoproteins as lectin chaperones. Approximately one-third of cellular proteins, including disease-related proteins, are synthesized in the ER. The lectin chaperones CRT and calnexin facilitate the correct folding of these glycoproteins; hence, these chaperones are essential for cells. Various CRT ligands have been reported, mainly composed of Glc1Man9GlcNAc2-type glycan. However, it remains problematic for the complicated synthesis and preparation, and it interacts with glycoprotein folding-related proteins in the ER other than CRT. This suggests that the development of CRT ligands still can be improved. In this study, we developed a hybrid binding concept, which encompasses concurrent binding of ligands to CRT lectin and chaperone domains. We synthesized a CRT-targeting glycan ligand with a glycan and hydrophobic aglycone for CRT lectin and chaperone domain binding, respectively. The thermal shift assay with the CRT-targeting glycan demonstrated that binding was enhanced by simultaneous glycan and hydrophobic aglycone binding. The affinity of the CRT-targeting ligand showed isothermal titration calorimetry approximately 50-fold stronger than that of the glycan alone, thereby supporting the hybrid binding concept. In addition, the CRT-targeting ligand inhibited chaperone function. Overall, these results indicate that the hybrid binding concept may be useful as a novel strategy for the development of CRT ligands and inhibitors.