Arata A, Sekiguchi M, Hirabayashi J, Kasai K
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Biol Pharm Bull. 2001 Jan;24(1):14-8. doi: 10.1248/bpb.24.14.
The 32-kDa galectin (LEC-1) of the nematode Caenorhabditis elegans (C elegans) is composed of two tandemly repeated homologous sequences, each containing a carbohydrate-recognition domain (CRD). Using the polymerase chain reaction (PCR) with LEC-1 cDNA as a template and "megaprimers", we performed site-directed mutagenesis to substitute conserved amino acid residues in these domains. The resultant mutated LEC-1s were produced in E. coli, and their binding abilities were estimated by affinity chromatography. When one of the conserved amino acid residues in the first lectin domain was substituted, the binding ability of the mutant protein to asialofetuin-agarose was reduced but still remained. The binding ability of such mutants was similar to that of the recombinant half molecule containing the second lectin domain (Ch). However, when mutations were introduced into the second lectin domain, the binding ability of these mutant lectins to asialofetuin-agarose was significantly reduced just like the half recombinant molecule containing the first lectin domain (Nh). The different effects of the substitution of amino acid residues on the two lectin domains suggest that the binding properties of the two sites are different and that LEC-1 acts as a "heterobifunctional crosslinker."
线虫秀丽隐杆线虫（C. elegans）的32 kDa半乳糖凝集素（LEC-1）由两个串联重复的同源序列组成，每个序列都包含一个碳水化合物识别结构域（CRD）。以LEC-1 cDNA为模板，使用“大引物”通过聚合酶链反应（PCR）进行定点诱变，以替换这些结构域中的保守氨基酸残基。将所得的突变型LEC-1在大肠杆菌中表达，并通过亲和色谱法评估其结合能力。当第一个凝集素结构域中的一个保守氨基酸残基被替换时，突变蛋白与去唾液酸胎球蛋白-琼脂糖的结合能力降低，但仍保留。这种突变体的结合能力与含有第二个凝集素结构域（Ch）的重组半分子相似。然而，当在第二个凝集素结构域中引入突变时，这些突变凝集素与去唾液酸胎球蛋白-琼脂糖的结合能力显著降低，就像含有第一个凝集素结构域（Nh）的重组半分子一样。氨基酸残基替换对两个凝集素结构域的不同影响表明，两个位点的结合特性不同，并且LEC-1起着“异双功能交联剂”的作用。
