NMR-based insight into galectin-3 binding to endothelial cell adhesion molecule CD146: Evidence for noncanonical interactions with the lectin’s CRD β-sandwich F-face
NMR-based insight into galectin-3 binding to endothelial cell adhesion molecule CD146: Evidence for noncanonical interactions with the lectin’s CRD β-sandwich F-face
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基于 NMR 的半乳糖凝集素 3 与内皮细胞粘附分子 CD146 结合的洞察：与凝集素 CRD β 三明治 F 面非典型相互作用的证据
DOI：
10.1093/glycob/cwz036
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发表时间：
2019
期刊：
影响因子：
4.3
通讯作者：
Kevin H Mayo
中科院分区：
文献类型：
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作者：
Zhongyu Zhang;Michelle C Miller;Xuejiao Xu;Chengcheng Song;Fan Zhang;Yi Zheng;Yifa Zhou;Guihua Tai;Kevin H Mayo
Galectin-3 (Gal-3) 与细胞粘附糖蛋白 CD146 结合，促进细胞因子分泌并介导内皮细胞迁移。在这里，我们使用核磁共振（NMR）15N-异核单量子相干（HSQC）光谱来研究 15N 标记的 Gal-3 与纯化 CD146 的胞外域（eFL）（五个 Ig 样胞外域 D1-D5）和较短的 D5 删除版本的 CD146（D1-D4）之间的结合。与 CD146 eFL 相比，Gal-3 及其碳水化合物识别域 (CRD) 与 CD146 D1–D4 的结合大大减少，支持了 D5 上存在更多糖基化位点的建议。尽管 Gal-3 β-三明治的典型糖结合 β-片 S-面（β-链 1、10、3、4、5、6）涉及与 CD146 的相互作用（例如 N-连接糖基化位点），但相对 Gal-3 F-面 β-片（β-链 11、2、7、图 8、9) 表明 Gal-3 F 面参与结合 CD146。观察结果支持了这一点，即添加乳糖虽然显着减弱 Gal-3 与 CD146 eFL 的结合（主要与 S 面结合），但并没有消除它。使用 Gal-3 F-face 突变体进行的生物层干涉测量研究得出的 KD 值表明，与野生型 Gal-3 相比，与 CD146 eFL 的净结合显着降低 (L203A) 或增加 (V204A、L218A、T243A)。然而，HSQC 乳糖滴定对糖与 Gal-3 CRD S-face 的结合没有显着影响。总体而言，我们的研究结果表明，Gal-3 与 CD146 的结合比与细胞受体上 β-半乳糖苷表位的简单相互作用更为复杂，并且凝集素的 CRD F 面在 CD146 结合过程中具有直接作用。
Galectin-3 (Gal-3) binds to cell adhesion glycoprotein CD146 to promote cytokine secretion and mediate endothelial cell migration. Here, we used Nuclear Magnetic Resonance (NMR)15N-Heteronuclear Single Quantum Coherence (HSQC) spectroscopy to investigate binding between15N-labeled Gal-3 and the extracellular domain (eFL) of purified CD146 (five Ig-like ectodomains D1–D5) and a shorter, D5-deleted version of CD146 (D1–D4). Binding of Gal-3 and its carbohydrate recognition domain (CRD) to CD146 D1–D4 is greatly reduced vis-à-vis CD146 eFL, supporting the proposal of a larger number of glycosylation sites on D5. Even though the canonical sugar-binding β-sheet S-face (β-strands 1, 10, 3, 4, 5, 6) of the Gal-3 β-sandwich is involved in interactions with CD146 (e.g. N-linked glycosylation sites), equivalent HSQC spectral perturbations at residues on the opposing Gal-3 F-face β-sheet (β-strands 11, 2, 7, 8, 9) indicate involvement of the Gal-3 F-face in binding CD146. This is supported by the observation that addition of lactose, while significantly attenuating Gal-3 binding (primarily with the S-face) to CD146 eFL, does not abolish it. Bio-Layer Interferometry studies with Gal-3 F-face mutants yield KDvalues to demonstrate a significant decrease (L203A) or increase (V204A, L218A, T243A) in net binding to CD146 eFL compared to wild type Gal-3. However, HSQC lactose titrations show no highly significant effects on sugar binding to the Gal-3 CRD S-face. Overall, our findings indicate that Gal-3 binding to CD146 is more involved than simple interactions with β-galactoside epitopes on the cell receptor, and that there is a direct role for the lectin’s CRD F-face in the CD146 binding process.