廣瀨 光了

Abstract Bacteriochlorophylls c and e are responsible for the main part of the light-harvesting process in chlorosome antenna systems of green sulfur bacteria and contain a methyl group at the peripheral C-20 position of their core chlorin rings. This study performed in vitro and in vivo analysis of the C-20 methyltransferase BchU derived from the green sulfur bacterium Chlorobaculum tepidum, which synthesizes bacteriochlorophyll c, to clarify the role of this enzyme in the biosynthetic pathway. Although the reaction step of BchU in the biosynthesis could not be determined by genetic analysis, enzymatic assays using various substrates showed that BchU reacts primarily with substrates after hydration of BchF and BchV at the C-3 position. The results in this study allow the proposition of a biosynthetic pathway for BChl c and e involving this enzyme.

Abstract 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.