Rinaldi Simon, Brennan Kathryn M, Goodyear Carl S, O'Leary Colin, Schiavo Giampietro, Crocker Paul R, Willison Hugh J
Division of Clinical Neurosciences, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8TA, UK.
Glycobiology. 2009 Jul;19(7):789-96. doi: 10.1093/glycob/cwp049. Epub 2009 Apr 6.
Glycolipids are major components of the plasma membrane, interacting with themselves, other lipids, and proteins to form an array of heterogeneous domains with diverse biological properties. Considerable effort has been focused on identifying protein binding partners for glycolipids and the glycan specificity for these interactions, largely achieved through assessing interactions between proteins and homogenous, single species glycolipid preparations. This approach risks overlooking both the enhancing and attenuating roles of heterogeneous glycolipid complexes in modulating lectin binding. Here we report a simple method for assessing lectin-glycolipid interactions. An automatic thin-layer chromatography sampler is employed to create easily reproducible arrays of glycolipids and their heterodimeric complexes immobilized on a synthetic polyvinyl-difluoride membrane. This array can then be probed with much smaller quantities of reagents than would be required using existing techniques such as ELISA and thin-layer chromatography with immuno-overlay. Using this protocol, we have established that the binding of bacterial toxins, lectins, and antibodies can each be attenuated, enhanced, or unaffected in the presence of glycolipid complexes, as compared with individual, isolated glycolipids. These findings underpin the wide-ranging influence and importance of glycolipid-glycolipid cis interactions when the nature of protein-carbohydrate recognition events is being assessed.
糖脂是质膜的主要成分，它们相互作用、与其他脂质以及蛋白质相互作用，形成一系列具有不同生物学特性的异质结构域。相当多的研究致力于确定糖脂的蛋白质结合伴侣以及这些相互作用的聚糖特异性，这主要是通过评估蛋白质与同质、单一物种糖脂制剂之间的相互作用来实现的。这种方法可能会忽视异质糖脂复合物在调节凝集素结合中的增强和减弱作用。在这里，我们报告一种评估凝集素 - 糖脂相互作用的简单方法。使用自动薄层色谱采样器在合成聚偏二氟乙烯膜上创建易于重现的糖脂及其异二聚体复合物阵列。然后，与使用现有技术（如酶联免疫吸附测定和免疫覆盖薄层色谱）相比，该阵列可以用更少的试剂进行检测。使用该方案，我们已经确定，与单个分离的糖脂相比，在糖脂复合物存在的情况下，细菌毒素、凝集素和抗体的结合可能会减弱、增强或不受影响。当评估蛋白质 - 碳水化合物识别事件的性质时，这些发现强调了糖脂 - 糖脂顺式相互作用的广泛影响和重要性。
