| Research Abstract |
X-ray crystallographic analyses of a Ca^<2+> dependent lectin CEL-I and its complex with carbohydrates were performed in order to elucidate its carbohydrate recognition mechanism. The results revealed that there is a Ca^<2+> ion located in the carbohydrate-binding site, and carbohydrates, such as N-acetylgalactosamine (GalNAc), was bound by coordinate as well as hydrogen bonds through 3-OH and 4-OH and Ca^<2+> and the four amino acid residues (Gin 101, Asp 103, Glu 109, Asn 123). In the case of CEL-I/GalNAc complex, there are additional hydrogen bonds between the acetoamido group of GalNAc and Arg 115, and also van der Waals interaction with Gln70 and the methyl group of the GalNAc. Therefore, these amino acid residues are replaced by alanine using site-directed mutagenesis technique to investigate the roles of these residues in GalNAc-recognition. As a result, substitution of Arg 115 reduced carbohydrate-binding activity to 1/8, while substitusion of Gln70 and Arg115 reduced it to abo
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ut 1/100. This proved importance of these residues in GalNAc-recognition, especially van der Waals contact of Gln70 and the methyl group of GalNAc seems very important.
On the other hand, crystal structure of CEL-III, Ca^<2+>-dependent hemolytic lectin in C. echinata, was also solved. The results indicated that this protein is composed of three domains (domains 1, 2, and 3), as had been expected from the amino acid sequence. Domains 1 and 2 have similar fold as the B-chains of toxic lectins ricin and abrin, and therefore these domains were thought to be carbohydrate-binding domains. However, in contrast to ricin and abrin, CEL-III contained seven Ca^<2+> in these domains, which are probably important to bind carbohydrates. Domain 3 of CEL-III showed novel structure rich in f3-strands. This domain contains hydrophobic region, which has been shown to exhibit antibacterial activity. Therefore, it is assumed that domain 3 is responsible for formation of cell membrane pores, after binding to cell-surface carbohydrates, leading to hemolysis. Less
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