Probing Heteromultivalent Protein-Glycosphingolipid Interactions using Native Mass Spectrometry and Nanodiscs
Probing Heteromultivalent Protein-Glycosphingolipid Interactions using Native Mass Spectrometry and Nanodiscs
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DOI：
10.1021/acs.analchem.9b05419
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发表时间：
2020-03-03
影响因子：
7.4
通讯作者：
Klassen, John S.
中科院分区：
文献类型：
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作者：
Han, Ling;Kitov, Pavel, I;Klassen, John S.
细胞表面的糖鞘糖脂(GSLS)和糖链结合蛋白(GBP)之间的相互作用介导了广泛的基本和病理过程。尽管这些相互作用具有重要的生物学意义，但大多数GBP的GSL配体仍有待确定，控制GSLS识别的机制也不完全清楚。最近，有人提出，当与高亲和力的配体一起存在时，低亲和力的GSL配体可以通过一种称为异多价结合的过程来显著促进GBP与多个结合位点的结合。在这里，为了直接确定异价GSL相互作用的存在并阐明其形成的机制，我们用天然质谱学和竞争性配基结合的方法研究了霍乱毒素B亚单位同五聚体(CTB5)与模型膜(纳米盘)中的神经节苷脂混合物的结合。电喷雾电离(ESI)-MS分析表明，纳米盘中高亲和力配体GM1(相对于结合部位的亚化学计量比)的存在促进了GD1b与CTB5的结合；在没有GM1的情况下，没有检测到GD1b结合。没有观察到CTB 5单独或与纳米盘中的GM1一起与其他五个被测神经节苷脂结合的直接ESI-MS证据。使用代理配体ESI-MS结合分析进行的亲和力测量证实，当与GM1一起存在时，GD1b与CTB5的结合显著增强(亲和力比GD1b寡糖亲和力高1000倍)。含有GM1和GM2、GD1a或GT1b的NDS也表现出增强CTB5结合的作用，但作用较小。在神经节苷脂纳米盘上进行的分子动力学模拟结果表明，低亲和力配体参与与GM1的异多价结合可能受神经节苷脂内部连接的Neu5Ac残基相对于膜表面的位置的调节。
Interactions between glycosphingolipids (GSLs) on the surfaces of cells and glycan-binding proteins (GBPs) mediate a wide variety of essential and pathological processes. Despite the biological importance of these interactions, the GSL ligands of most GBPs remain to be identified and the mechanisms controlling recognition of GSLs are incompletely understood. Recently, it was suggested that, when present together with high affinity ligands, low affinity GSL ligands can contribute significantly to the binding of GBPs with multiple binding sites through a process called heteromultivalent binding. Here, with goal of directly establishing the existence of heteromultivalent GSL interactions and elucidating the mechanism underlying their formation, we investigated cholera toxin B subunit homopentamer (CTB5) binding to ganglioside mixtures in model membranes (nanodiscs) using native mass spectrometry (MS) and competitive ligand binding. Electrospray ionization (ESI)-MS analysis revealed that the presence of the high affinity ligand GM1 (at substoichiometric amounts relative to binding sites) in the nanodisc promotes GD1b binding to CTB5; no GD1b binding was detected in the absence of GM1. No direct ESI-MS evidence of CTB 5 binding to the other five gangliosides tested, alone or present together with GM1 in the nanodiscs, was observed. Affinity measurements, carried out using the proxy ligand ESI-MS binding assay, confirmed that GD1b binding to CTB5 is dramatically enhanced (>1000-times higher affinity compared to the GD1b oligosaccharide affinity) when present with GM1. NDs containing GM1 and GM2, GD1a, or GT1b also exhibited enhanced CTB5 binding, however, the effect was smaller. The results of molecular dynamics simulations performed on ganglioside-containing nanodiscs suggest that the participation of low affinity ligands in heteromultivalent binding with GM1 may be regulated by the positions of the internal Gal-linked Neu5Ac residues of the gangliosides relative to the membrane surface.