Summary Proteins containing DM9 motifs, which were originally identified in the Drosophila melanogaster genome, are widely distributed in various organisms and are assumed to be involved in their innate immune response. In this study, we produced a recombinant protein of CG13321 (rCG13321) from D. melanogaster, which consists of four DM9 motifs, in Escherichia coli cells. In affinity chromatography using a mannose-immobilized column, rCG13321 exhibited mannose-binding ability and was separated into high-affinity and low-affinity fractions, named HA and LA, respectively, based on its binding ability to the column. In addition to having a higher affinity for the column, HA exhibited self-oligomerization ability, suggesting slight differences in tertiary structure. Both LA and HA showed hemagglutinating activity and were able to agglutinate an oligomannose-containing dendrimer, indicating that they have multiple carbohydrate-binding sites. Glycan array analysis suggested that rCG13321 primarily recognizes D-mannose and D-rhamnose through hydrogen bonding with the 2-, 3-, and 4-hydroxy groups. Isothermal titration calorimetry demonstrated that rCG13321 has a comparable affinity to typical lectins. These findings suggest that CG13321 functions as a carbohydrate-binding protein or lectin that recognizes mannose and related carbohydrate-containing molecules on the surface of foreign organisms as a pattern recognition molecule.

中文翻译：

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含有黑腹果蝇 DM9 基序的重组蛋白的碳水化合物结合能力

摘要 含有 DM9 基序的蛋白质最初是在果蝇基因组中发现的，广泛分布于各种生物体中，并被认为参与其先天免疫反应。在本研究中，我们在大肠杆菌细胞中从黑腹果蝇中产生了 CG13321 (rCG13321) 重组蛋白，该蛋白由四个 DM9 基序组成。在使用甘露糖固定柱的亲和层析中，rCG13321表现出甘露糖结合能力，并根据其与柱的结合能力分为高亲和力和低亲和力级分，分别命名为HA和LA。除了对柱具有较高的亲和力外，HA 还表现出自寡聚能力，表明三级结构略有差异。 LA 和 HA 均显示出血凝活性，并且能够凝集含有寡聚甘露糖的树枝状聚合物，表明它们具有多个碳水化合物结合位点。聚糖阵列分析表明，rCG13321 主要通过与 2-、3-和 4-羟基形成氢键来识别 D-甘露糖和 D-鼠李糖。等温滴定量热法证明 rCG13321 与典型凝集素具有相当的亲和力。这些发现表明，CG13321 作为碳水化合物结合蛋白或凝集素发挥作用，将外来生物体表面上的甘露糖和相关含碳水化合物分子识别为模式识别分子。