Analyses of carbohydrate-recognition mechanism of calcium-dependent lectins and its application to construct novel functional proteins

本文对Ca^<2+>依赖性凝集素cel - 1及其与碳水化合物的复合物进行了x射线晶体学分析，以阐明其碳水化合物识别机制。结果表明，碳水化合物结合位点存在一个Ca^<2+ b>离子，碳水化合物如n -乙酰半乳糖胺（GalNAc）通过3-OH、4-OH、Ca^<2+>和4个氨基酸残基（Gin 101、Asp 103、Glu 109、Asn 123）通过配位键和氢键结合。在cel - 1 /GalNAc配合物中，GalNAc的乙酰氨基与Arg 115之间存在额外的氢键，并且与Gln70和GalNAc的甲基之间存在范德华作用。因此，这些氨基酸残基被丙氨酸取代，使用定点诱变技术来研究这些残基在galnac识别中的作用。结果表明，Arg115取代后的碳水化合物结合活性降至1/8，而Gln70和Arg115取代后的碳水化合物结合活性降至1/100以上。这证明了这些残基在GalNAc识别中的重要性，特别是Gln70的范德华接触和GalNAc的甲基似乎非常重要。另一方面，还解决了C. chinata中Ca^<2+>-依赖性溶血凝集素cell - iii的晶体结构。结果表明，该蛋白由3个结构域（结构域1、2和3）组成，与氨基酸序列一致。结构域1和2与有毒凝集素蓖麻毒素和蓖麻毒素的b链具有相似的折叠，因此这些结构域被认为是碳水化合物结合结构域。然而，与蓖麻毒素和蓖麻毒素相比，CEL-III在这些结构域中含有7个Ca^<2+>，这可能对结合碳水化合物很重要。CEL-III的结构域3显示出富含f3链的新颖结构。该结构域含有疏水区，已被证明具有抗菌活性。因此，假设结构域3与细胞表面碳水化合物结合后，负责细胞膜孔的形成，导致溶血。少

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 … More 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