Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University , S-106 91 Stockholm, Sweden.
Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University , S-106 91 Stockholm, Sweden.
J Am Chem Soc. 2017 Jan 11;139(1):218-230. doi: 10.1021/jacs.6b09534. Epub 2016 Dec 27.
The highly poisonous botulinum neurotoxins, produced by the bacterium Clostridium botulinum, act on their hosts by a high-affinity association to two receptors on neuronal cell surfaces as the first step of invasion. The glycan motifs of gangliosides serve as initial coreceptors for these protein complexes, whereby a membrane protein receptor is bound. Herein we set out to characterize the carbohydrate minimal binding epitope of the botulinum neurotoxin serotype A. By means of ligand-based NMR spectroscopy, X-ray crystallography, computer simulations, and isothermal titration calorimetry, a screening of ganglioside analogues together with a detailed characterization of various carbohydrate ligand complexes with the toxin were accomplished. We show that the representation of the glycan epitope to the protein affects the details of binding. Notably, both branches of the oligosaccharide GD1a can associate to botulinum neurotoxin serotype A when expressed as individual trisaccharides. It is, however, the terminal branch of GD1a as well as this trisaccharide motif alone, corresponding to the sialyl-Thomsen-Friedenreich antigen, that represents the active ligand epitope, and these compounds bind to the neurotoxin with a high degree of predisposition but with low affinities. This finding does not correlate with the oligosaccharide moieties having a strong contribution to the total affinity, which was expected to be the case. We here propose that the glycan part of the ganglioside receptors mainly provides abundance and specificity, whereas the interaction with the membrane itself and protein receptor brings about the strong total binding of the toxin to the neuronal membrane.
由肉毒梭菌产生的剧毒肉毒神经毒素，通过与神经元细胞表面上的两个受体的高亲和力结合，作为入侵的第一步作用于其宿主。神经节苷脂的糖基结构作为这些蛋白复合物的初始核心受体，其中膜蛋白受体被结合。在此，我们着手研究 A 型肉毒神经毒素的碳水化合物最小结合表位。通过基于配体的 NMR 光谱学、X 射线晶体学、计算机模拟和等温滴定量热法，对神经节苷脂类似物进行了筛选，并对各种与毒素结合的碳水化合物配体复合物进行了详细表征。我们表明，糖基表位对蛋白质的呈现方式影响结合的细节。值得注意的是，当表达为单个三糖时，GD1a 聚糖的两个分支都可以与 A 型肉毒神经毒素结合。然而，正是 GD1a 的末端分支以及单独的这个三糖基序，对应于唾液酸-Thomsen-Friedenreich 抗原，代表了活性配体表位，这些化合物与神经毒素具有高度的倾向性结合，但亲和力较低。这一发现与预期的情况不符，即寡糖部分对总亲和力有很强的贡献。我们在此提出，神经节苷脂受体的糖部分主要提供丰度和特异性，而与膜本身和蛋白受体的相互作用导致毒素与神经元膜的强总结合。
