|Budget Amount *help
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2006: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2005: ¥2,500,000 (Direct Cost: ¥2,500,000)
In this study, we have analyzed the carbohydrate-binding mechanism of Ca^<2+>-dependent lectins, especially C.echinata lectins CEL-I, CEL-I II, and CEL-IV to obtain information to design novel proteins with various carbohydrate-recognition abilities. Among these lectins, CEL-I has extremely high specificity for N-acetylgalactosamine (GalNAc), and several site-directed mutants of it were prepared. As a result, Gln70 and Arg115, especially Gln70 was found to be important for high specificity for GalNAc. In addition, it was also shown that mutations to Arg115 lead to decreased GalNAc-specificity of this lectin. On the other hand, Trp79 of CEL-IV, which has been shown to be involved in the carbohydrate-binding site based on the x-ray crystallographic analysis, was mutated to Tyr, His, and Ala to examine the effects of aromatic residues in interaction with carbohydrate. The results revealed that the affinity of mutants for GalNAc decreased in the order of Trp, Tyr, His, and Ala mutations, indicating that interaction between bulky aromatic side chains in the carbohydrate-binding site is important for stabilization of binding of the specific carbohydrates. We have also prepared mutants of domain 3 and its α-helix region of the hemolytic lectin CEL-III as glutathione S-transferase (GST)-fusion proteins to elucidate the role of this domain in the oligomerization process in the target cell membranes. These fusion proteins exhibited self-oligomerization ability when expressed in E.coli cells. However, when the characteristic Val residues were mutated to Ala residues, the oligomerization ability markedly decreased with increasing the number of Ala-mutations. These results demonstrated that the α-helix region, especially its Val-clusters, of domain 3 plays a crucial role in self-oligomerization of CEL-III in the target cell membranes.