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

从长鳗的皮肤黏液中鉴定出两种凝集素，分别称为凝集素1和凝集素2 （Con1和Con2）。它们具有48%的序列同源性和不同的碳水化合物结合特异性。为了分析集合体的详细结构和碳水化合物结合能力的关系，本研究通过位点直接诱变制备了位于外周碳水化合物结合间隙的氨基酸残基上的Con1和Con2的互变取代突变体。利用正面亲和色谱系统分析了它们对34种吡啶层合寡糖的结合特异性，发现它们具有独特的糖特异性。例如，发现Congerins的V68N、Q71E、Q72T、E74Q和T107N突变影响了较强的碳水化合物结合活性。另一方面，已知Congerins对人T细胞系Jurkat细胞的凋亡细胞死亡具有较强的诱导活性。本研究利用Jurkat细胞检测了Congerin突变体的细胞毒活性，以评价其生物活性。比较各突变体的ED50，可以发现Congerin突变体的凋亡活性与糖结合活性之间存在明显的相关性，提示特异性的糖识别对凋亡活性有重要影响。因此，已经阐明了Congerin突变体获得独特的糖结合谱，可作为分析独特碳水化合物结构和活性的工具。

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.