Siglec-7 is a human CD33-like siglec, and is localised predominantly on human natural killer (NK) cells and monocytes. Siglec-7 is considered to function as an immunoreceptor in a sialic acid-dependent manner. However, the underlying mechanisms linking sialic acid-binding and function remain unknown. Here, to gain new insights into the ligand-binding properties of Siglec-7, we carried out in silico analysis and site-directed mutagenesis, and found a new sialic acid-binding region (site 2 containing R67) in addition to the well-known primary ligand-binding region (site 1 containing R124). This was supported by equilibrium dialysis, STD-NMR experiments, and inhibition analysis of GD3-binding toward Siglec-7 using synthetic sialoglycoconjugates and a comprehensive set of ganglioside-based glycoconjugates. Our results suggest that the two ligand-binding sites are potentially controlled by each other due to the flexible conformation of the C-C′ loop of Siglec-7.

Siglec-7是人类CD33样的siglec，主要位于人类自然杀伤（NK）细胞和单核细胞上。Siglec-7被认为以唾液酸依赖性方式起免疫受体的作用。然而，连接唾液酸结合和功能的潜在机制仍然未知。在这里，为了获得对Siglec-7的配体结合特性的新见解，我们进行了计算机模拟进行了分析和定点诱变，并发现了一个新的唾液酸结合区（含有R67的位点2）以及著名的一级配体结合区（含有R124的位点1）。平衡透析，STD-NMR实验以及使用合成唾液酸糖共轭物和一整套基于神经节苷脂的糖共轭物对GD3与Siglec-7的结合的抑制分析均支持了这一点。我们的结果表明，由于Siglec-7的CC'环具有柔性构象，因此两个配体结合位点可能相互控制。