While there are many methods to quantify the synthesis, localization, and pool sizes of proteins and DNA during physiological responses and toxicological stress, only few approaches allow following the fate of carbohydrates. One of them is metabolic glycoengineering (MGE), which makes use of chemically modified sugars (CMS) that enter the cellular biosynthesis pathways leading to glycoproteins and glycolipids. The CMS can subsequently be coupled (via bio-orthogonal chemical reactions) to tags that are quantifiable by microscopic imaging. We asked here, whether MGE can be used in a quantitative and time-resolved way to study neuronal glycoprotein synthesis and its impairment. We focused on the detection of sialic acid (Sia), by feeding human neurons the biosynthetic precursor N-acetyl-mannosamine, modified by an azide tag. Using this system, we identified non-toxic conditions that allowed live cell labeling with high spatial and temporal resolution, as well as the quantification of cell surface Sia. Using combinations of immunostaining, chromatography, and western blotting, we quantified the percentage of cellular label incorporation and effects on glycoproteins such as polysialylated neural cell adhesion molecule. A specific imaging algorithm was used to quantify Sia incorporation into neuronal projections, as potential measure of complex cell function in toxicological studies. When various toxicants were studied, we identified a subgroup (mitochondrial respiration inhibitors) that affected neurite glycan levels several hours before any other viability parameter was affected. The MGE-based neurotoxicity assay, thus allowed the identification of subtle impairments of neurochemical function with very high sensitivity.

中文翻译：

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时间和空间分辨的质膜唾液酸化定量，用于测量细胞功能和神经毒性。

尽管有很多方法可以量化生理反应和毒理学过程中蛋白质和DNA的合成，定位和合并大小，但只有很少的方法可以追踪碳水化合物的命运。其中之一是代谢糖工程（MGE），它利用化学修饰的糖（CMS）进入细胞的生物合成途径，从而产生糖蛋白和糖脂。CMS随后可以（通过生物正交化学反应）耦合到可通过显微镜成像量化的标签。我们在这里问，是否可以以定量和时间分辨的方式使用MGE来研究神经元糖蛋白的合成及其损伤。我们专注于唾液酸（Sia）的检测，方法是向人类神经元提供经过叠氮化物标签修饰的生物合成前体N-乙酰基甘露糖胺。使用这个系统，我们确定了无毒条件，可以以高时空分辨率对活细胞进行标记，并对细胞表面Sia进行定量。使用免疫染色，色谱和蛋白质印迹的组合，我们量化了细胞标记掺入的百分比和对糖蛋白如多唾液酸化神经细胞粘附分子的影响。使用一种特殊的成像算法来量化Sia掺入神经元预测中的能力，作为毒理学研究中复杂细胞功能的潜在量度。在研究了各种有毒物质后，我们确定了一个亚组（线粒体呼吸抑制剂），该亚组在其他任何生存力参数受到影响之前数小时就影响了神经突聚糖水平。基于MGE的神经毒性测定，