Clarification of Carbohydrate-Recognition Mechanisms of Gralectins which Evolved via Accelerated Substitution and Their Application for Biotechnology

从海鳗皮肤粘液中分离到两种半乳糖凝集素，分别命名为Congerin I和Congerin II（Con1和Con2）。它们显示出48%的序列同一性，具有不同的碳水化合物结合特异性。为了分析Con1和Con2的详细结构和糖结合能力之间的关系，本研究采用定点突变的方法，在Con1和Con2的外周糖结合裂隙的氨基酸残基上制备了相互取代的突变体。利用正面亲和层析系统分析了它们对34种吡啶胺寡糖的结合特异性，结果表明它们具有独特的糖特异性。例如，发现Congerins的V68N、Q71E、Q72T、E74Q和T107N突变影响强碳水化合物结合活性。另一方面，已知Congerins对人T细胞系Jurkat细胞的凋亡性细胞死亡具有强的诱导活性。在这项研究中，用Jurkat细胞检测Congerin突变体的细胞毒活性，以评估其生物学活性。通过比较各突变体的ED 50，发现Congerin突变体的细胞凋亡活性与糖结合活性之间存在明显的相关性，表明糖的特异性识别对细胞凋亡活性具有重要作用。因此，已经阐明了获得独特糖结合谱的Congerin突变体可用作分析独特碳水化合物的结构和活性的工具。

Two galectins termed Congerin I and Congerin II (Con1 and Con2) have been identified from skin mucus of conger eel. They showed 48% sequence identity with different carbohydrate-binding specificity. To analysis the detailed structure and carbohydrate binding ability relationships of congeries, in this study, reciprocal substituted mutants between Con1 and Con2 at the amino acid residues located in peripheral carbohydrate-binding clefts were prepared by site-direct mutagenesis. Analyses of their binding-specificity against 34 kinds of pyridylaminated oligosaccharides using frontal affinity chromatography system revealed that they had unique sugar specificities. For examples, it was found that V68N, Q71E, Q72T, E74Q and T107N mutations of Congerins affected on the strong carbohydrate binding activity. On the other hand, Congerins have already known had strong inducing activity for apoptotic cell death of human T cell lines, Jurkat cells. In this study, the cytotoxic activities of Congerin mutants were assayed using Jurkat cells to evaluate their biological activities. Comparison of ED50 of each mutant showed clearly correlations between apoptotic activities and sugar binding activities of Congerin mutants, suggesting that the specific sugar recognition was important for the apoptotic activity. Thus, it has been elucidated that the Congerin mutants obtained unique sugar binding profiles are applicable as tools for analyzing the structure and activities of unique carbohydrates.