Alberta Glycomics Centre and Department of Chemistry, University of Alberta , Edmonton, Alberta, Canada T6G 2G2.
Department of Pharmacology, University of Alberta , Edmonton, Alberta, Canada T6G 2H7.
Anal Chem. 2017 Sep 5;89(17):9330-9338. doi: 10.1021/acs.analchem.7b02094. Epub 2017 Aug 17.
This work describes a versatile analytical approach, which combines the proxy ligand electrospray ionization mass spectrometry (ESI-MS) assay and model membranes of defined composition, to quantify the influence of lipid bilayer composition on protein-glycolipid binding in vitro. To illustrate the implementation of the assay (experimental design and data analysis), affinities of the monosialoganglioside ligand GM1, incorporated into nanodiscs (NDs), for cholera toxin B subunit homopentamer (CTB) were measured. A series of NDs containing GM1 and cholesterol were prepared using three different phospholipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)), and the average GM1 and cholesterol content of each ND were determined. The intrinsic affinities of GM1-containing NDs prepared with the three phospholipids are found to be similar in magnitude, indicating that small differences in the fatty acid chain length and the number of unsaturated bonds do not significantly affect the CTB-GM1 interaction. Moreover, the measured affinities are similar to the value measured for GM1 pentasaccharide, indicating that neither the ceramide moiety nor the surface of the phospholipid membrane plays a significant role in CTB binding. The intrinsic (per binding site) affinity of the CTB-GM1 interaction was found to decrease with increasing GM1 content of the ND, consistent with the occurrence of GM1 clustering in the membrane, which sterically hinders binding to CTB. Notably, the addition of cholesterol to GM1-containing NDs did not have a significant effect on the strength of the CTB-GM1 interaction. This result, which is at odds with the findings of a previous study of CTB binding to GM1 in vesicles, suggests that cholesterol does not "mask" GM1, at least not in NDs. These data, in addition to providing new insights into the influence of membrane composition on CTB-GM1 binding, demonstrate the potential of the proxy ligand ESI-MS approach for comprehensive and quantitative studies of lectin interactions with glycolipids in native-like, membrane environments.
这项工作描述了一种多功能的分析方法，该方法结合了配体代用电喷雾电离质谱（ESI-MS）测定法和具有明确组成的模型膜，以定量研究脂质双层组成对体外蛋白-糖脂结合的影响。为了说明该测定法的实施（实验设计和数据分析），我们测量了单唾液酸神经节苷脂配体 GM1 与霍乱毒素 B 亚基五聚体（CTB）的结合亲和力，GM1 被包封在纳米盘（NDs）中。使用三种不同的磷脂（1,2-二肉豆蔻酰基-sn-甘油-3-磷酸胆碱（DMPC）、1,2-二月桂酰基-sn-甘油-3-磷酸胆碱（DPPC）和 1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱（POPC））制备了一系列含有 GM1 和胆固醇的 NDs，并确定了每个 ND 中的 GM1 和胆固醇的平均含量。发现用三种磷脂制备的含 GM1 的 NDs 的固有亲和力在数量级上相似，表明脂肪酸链长和不饱和度的小差异不会显著影响 CTB-GM1 相互作用。此外，测量得到的亲和力与 GM1 五糖测量得到的亲和力相似，表明神经酰胺部分或磷脂膜的表面都没有在 CTB 结合中发挥重要作用。发现 CTB-GM1 相互作用的固有（每个结合位）亲和力随 ND 中 GM1 含量的增加而降低，这与膜中 GM1 聚集一致，GM1 聚集会阻碍 CTB 的结合。值得注意的是，向含有 GM1 的 ND 中添加胆固醇对 CTB-GM1 相互作用的强度没有显著影响。这一结果与先前关于 CTB 与囊泡中 GM1 结合的研究结果不一致，表明胆固醇不会“掩盖”GM1，至少在 ND 中不会。这些数据除了提供关于膜组成对 CTB-GM1 结合影响的新见解外，还表明配体代用 ESI-MS 方法具有用于全面和定量研究在天然样膜环境中凝集素与糖脂相互作用的潜力。
