Department of Chemistry, University of Alberta, Edmonton T6G 2G2, Alberta, Canada.
Department of Biochemistry, University of Alberta, Edmonton T6G 2R3, Canada.
Anal Chem. 2022 Nov 22;94(46):16042-16049. doi: 10.1021/acs.analchem.2c03067. Epub 2022 Nov 11.
Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) are involved in numerous physiological and pathophysiological processes. Many model membrane systems are available for studying GBP-GSL interactions, but a systematic investigation has not been carried out on how the nature of the model membrane affects binding. In this work, we use electrospray ionization mass spectrometry (ESI-MS), both direct and competitive assays, to measure the binding of cholera toxin B subunit homopentamer (CTB) to GM1 ganglioside in liposomes, bilayer islands [styrene maleic acid lipid particles (SMALPs), nanodiscs (NDs), and picodiscs (PDs)], and micelles. We find that direct ESI-MS analysis of CTB binding to GM1 is unreliable due to non-uniform response factors, incomplete extraction of bound GM1 in the gas phase, and nonspecific CTB-GM1 interactions. Conversely, indirect proxy ligand ESI-MS measurements show that the intrinsic (per binding site) association constants of CTB for PDs, NDs, and SMALPs are similar and comparable to the affinity of soluble GM1 pentasaccharide (GM1). The observed affinity decreases with increasing GM1 content due to molecular crowding stemming from GM1 clustering. Unlike the smaller model membranes, the observed affinity of CTB toward GM1 liposomes is ∼10-fold weaker than GM1 and relatively insensitive to the GM1 content. GM1 glycomicelles exhibit the lowest affinity, ∼35-fold weaker than GM1. Together, the results highlight experimental design considerations for quantitative GBP-GSL binding studies involving multisubunit GBPs and factors to consider when comparing results obtained with different membrane systems. Notably, they suggest that bilayer islands with a low percentage of GSL, wherein clustering is minimized, are ideal for assessing intrinsic strength of GBP-GSL interactions in a membrane environment, while binding to liposomes, which is sub-optimal due to extensive clustering, may be more representative of authentic cellular environments.
糖基结合蛋白 (GBP) 与糖脂 (GSL) 之间的相互作用涉及许多生理和病理生理过程。有许多模型膜系统可用于研究 GBP-GSL 相互作用，但尚未系统地研究模型膜的性质如何影响结合。在这项工作中，我们使用电喷雾电离质谱 (ESI-MS)，包括直接和竞争性测定，来测量霍乱毒素 B 亚基五聚体 (CTB) 与 GM1 神经节苷脂在脂质体、双层岛 [苯乙烯马来酸脂颗粒 (SMALPs)、纳米盘 (NDs) 和皮克盘 (PDs)] 和胶束中的结合。我们发现，由于不均匀的响应因子、气相中结合的 GM1 不完全提取以及非特异性 CTB-GM1 相互作用，直接 ESI-MS 分析 CTB 与 GM1 的结合是不可靠的。相反，间接配体 ESI-MS 测量表明，CTB 与 PDs、NDs 和 SMALPs 的内在（每个结合位点）缔合常数相似且可与可溶性 GM1 五糖 (GM1) 的亲和力相媲美。由于 GM1 聚集引起的分子拥挤，观察到的亲和力随 GM1 含量的增加而降低。与较小的模型膜不同，观察到的 CTB 对 GM1 脂质体的亲和力比 GM1 弱约 10 倍，并且对 GM1 含量相对不敏感。GM1 糖脂体表现出最低的亲和力，比 GM1 弱约 35 倍。总的来说，这些结果强调了涉及多亚基 GBP 和糖脂相互作用的定量 GBP-GSL 结合研究的实验设计注意事项，以及在比较不同膜系统获得的结果时需要考虑的因素。值得注意的是，它们表明，在膜环境中评估 GBP-GSL 相互作用的固有强度时，低百分比 GSL 的双层岛（其中聚类最小）是理想的，而由于广泛聚类导致结合到脂质体是不理想的，这可能更能代表真实的细胞环境。
