Wratil Paul R, Horstkorte Rüdiger
Max von Pettenkofer-Institut & Genzentrum, Virologie, Nationales Referenzzentrum für Retroviren, Medizinische Fakultät, LMU München; Institut für Laboratoriumsmedizin, klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin.
Institut für Physiologische Chemie, Martin-Luther-Universität Halle-Wittenberg;
J Vis Exp. 2017 Nov 25(129):55746. doi: 10.3791/55746.
Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.
唾液酸（Sia）是糖缀合物（如N - 和O - 聚糖或糖脂）的极其重要的组成部分。由于其位于寡糖和多糖的非还原末端的位置，以及其独特的化学特性，唾液酸参与了多种不同的受体 - 配体相互作用。通过改变细胞表面唾液酸的表达，唾液酸依赖性相互作用将因此受到影响。这有助于研究唾液酸依赖性相互作用，并有可能以有益的方式影响某些疾病。通过代谢糖工程（MGE），可以调节细胞表面唾液酸的表达。在此，用N - 乙酰甘露糖胺（ManNAc）的C2修饰衍生物处理细胞、组织甚至整个动物。这些氨基糖作为唾液酸前体分子，因此被代谢为相应的唾液酸种类并在糖缀合物上表达。应用该方法对各种生物过程产生有趣的影响。例如，它可以大幅降低处理过的神经元细胞中多唾液酸（polySia）的表达，从而影响神经元的生长和分化。在这里，我们展示了两种最常见的C2修饰的N - 酰基甘露糖胺衍生物，N - 丙酰甘露糖胺（ManNProp）以及N - 丁酰甘露糖胺（ManNBut）的化学合成，并进一步展示了这些非天然氨基糖如何应用于细胞培养实验。通过高效液相色谱（HPLC）对修饰的唾液酸种类的表达进行定量，并通过质谱进一步分析。使用市售的多唾液酸抗体通过蛋白质印迹法阐明对多唾液酸表达的影响。
