Human sialic-acid-binding immunoglobulin-like lectin-9 (Siglec-9) is a glycoimmune checkpoint receptor expressed on several immune cells. Binding of Siglec-9 to sialic acid containing glycans (sialoglycans) is well documented to modulate its functions as an inhibitory receptor. Here, we first assigned the amino acid backbone of the Siglec-9 V-set domain (Siglec-9_d1), using well-established triple resonance three-dimensional nuclear magnetic resonance (NMR) methods. Then, we combined solution NMR and molecular dynamic simulation methods to decipher the molecular details of the interaction of Siglec-9 with the natural ligands α2,3 and α2,6 sialyl lactosamines (SLN), sialyl Lewis X (sLeX), and 6-O sulfated sLeX and with two synthetically modified sialoglycans that bind with high affinity. As expected, Neu5Ac is accommodated between the F and G β-strands at the canonical sialic acid binding site. Addition of a heteroaromatic scaffold 9N-5-(2-methylthiazol-4-yl)thiophene sulfonamide (MTTS) at the C9 position of Neu5Ac generates new interactions with the hydrophobic residues located at the G–G′ loop and the N-terminal region of Siglec-9. Similarly, the addition of the aromatic substituent (5-N-(1-benzhydryl-1H-1,2,3-triazol-4-yl)methyl (BTC)) at the C5 position of Neu5Ac stabilizes the conformation of the long and flexible B′–C loop present in Siglec-9. These results expose the underlying mechanism responsible for the enhanced affinity and specificity for Siglec-9 for these two modified sialoglycans and sheds light on the rational design of the next generation of modified sialoglycans targeting Siglec-9.

中文翻译：

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通过 NMR 波谱和分子动力学建模揭示 Siglec-9 对聚糖配体的分子识别

人唾液酸结合免疫球蛋白样凝集素 9 (Siglec-9) 是一种在多种免疫细胞上表达的糖免疫检查点受体。 Siglec-9 与含有唾液酸的聚糖（唾液酸聚糖）的结合已被充分证明可以调节其作为抑制性受体的功能。在这里，我们首先使用成熟的三重共振三维核磁共振 (NMR) 方法指定了 Siglec-9 V-set 结构域 (Siglec-9 _d1 )的氨基酸主链。然后，我们结合溶液核磁共振和分子动力学模拟方法，破译了 Siglec-9 与天然配体 α2,3 和 α2,6 唾液酸乳糖胺 (SLN)、唾液酸路易斯 X (sLeX) 和 6- 相互作用的分子细节。 O 硫酸化 sLeX 并具有两种以高亲和力结合的合成修饰唾液聚糖。正如预期的那样，Neu5Ac 位于 F 和 G β 链之间的典型唾液酸结合位点处。在 Neu5Ac 的 C9 位置添加杂芳族支架 9 N -5-(2-甲基噻唑-4-基)噻吩磺酰胺 (MTTS)，与位于 G-G' 环和 N 末端的疏水残基产生新的相互作用Siglec-9 区域。类似地，在 Neu5Ac 的 C5 位上添加芳香族取代基 (5- N- (1-二苯甲基-1H- 1,2,3-三唑-4-基)甲基 (BTC)) 可稳定长链的构象。 Siglec-9 中存在灵活的 B′-C 环。这些结果揭示了 Siglec-9 对这两种修饰唾液酸聚糖的亲和力和特异性增强的潜在机制，并揭示了针对 Siglec-9 的下一代修饰唾液酸聚糖的合理设计。