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

钙依赖性凝集素碳水化合物识别机制分析及其在新型功能蛋白构建中的应用

基本信息

批准号：
14560073
负责人：
HATAKEYAMA Tomomitsu
金额：
$ 2.3万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

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

为了阐明其碳氢化物识别机制，进行了Ca^<2+>依赖性讲座及其与碳水合物的复合物的X射线晶体学分析。 The results revealed that there is a Ca^<2+> ion located in the carbonhydrate-binding site, and carbonhydrates, 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).在Cel-I/GalNAC复合物的情况下，Galnac和Arg 115的乙酰氨酰胺组之间还有其他氢键，以及范德华与Gln70和Galnac的甲基相互作用。因此，这些氨基酸残基被丙氨酸用位于定位的诱变技术取代，以研究这些残基在GalNAC识别中的作用。结果，ARG 115的替代碳水化合物结合活性降低至1/8，而替代GLN70和ARG115则将其降低为ABO…更多UT 1/100。这些残留物在GalNAC识别中被证明是重要的，尤其是GLN70的范德华接触和GalNAC的甲基接触似乎非常重要。另一方面，在C. echinata中的Cel-III的晶体结构，Ca^<2+> - 依赖性溶血讲座的晶体结构也得到了解决。结果表明该蛋白质由三个域（结构域1、2和3）组成，如氨基酸序列所预期的那样。域1和2具有与有毒凝集素的ricin和abrin的B链相似的折叠，因此这些结构域被认为是碳水化合物结合域。然而，与ricin和abrin相反，Cel-III在这些结构域中包含七个Ca^<2+>，这对于结合碳水性可能很重要。 Cel-III的域3显示了具有F3链的新型结构。该结构域包含疏水区，已显示出暴露于抗菌活性。因此，假定结构域3在与细胞表面碳液结合后导致细胞膜孔的形成，从而导致溶血。较少的