Péanne Romain, Vanbeselaere Jorick, Vicogne Dorothée, Mir Anne-Marie, Biot Christophe, Matthijs Gert, Guérardel Yann, Foulquier François
Center for Human Genetics, KU Leuven University, Herestraat 49, Leuven, Belgium.
Methods Cell Biol. 2013;118:157-76. doi: 10.1016/B978-0-12-417164-0.00010-0.
Modifications of N-glycosylation in disease states are common and illustrate the crucial requirement of glycosylation in human biology. Mainly based on glycan permethylation and the use of mass spectrometry analysis, we can easily understand that many different methods to analyze the N-glycome have seen the day. While extremely powerful, these methods are mainly used to analyze qualitative variations of N-glycosylation of human serum proteins and do not necessarily reflect the glycosylation status of derived mammalian cultured cells. This chapter summarizes two methods that we are routinely using in our laboratory to assess the ER and Golgi N-glycosylation process. The proposed methodology allows pinpointing ER as well as Golgi glycosylation deficiencies in mammalian cultured cells. The first approach is based on direct metabolic labeling of cultured mammalian cells with [2-(3)H] mannose followed by sequential extraction and HPLC analysis of the purified oligosaccharides. The second one is based on the copper-catalyzed azide alkyne cycloaddition (CuAAC) strategy. We propose the use of alkyne-tagged sialic acid (SialNAl) to visualize the Golgi glycosylation efficiency. Their metabolic incorporation into newly synthesized glycoproteins can then be chemoselectively coupled to complementary azide-functionalized fluorophores, and visualized by using confocal laser scanning microscopy. To summarize, we present here a detailed description of our know-how in the field of ER and Golgi N-glycosylation.
疾病状态下N-糖基化的修饰很常见，这表明糖基化在人类生物学中至关重要。主要基于聚糖全甲基化和质谱分析的应用，我们可以很容易地了解到，许多不同的分析N-糖组的方法已经出现。虽然这些方法非常强大，但主要用于分析人血清蛋白N-糖基化的定性变化，并不一定反映衍生的哺乳动物培养细胞的糖基化状态。本章总结了我们实验室常规使用的两种评估内质网和高尔基体N-糖基化过程的方法。所提出的方法可以确定哺乳动物培养细胞中的内质网以及高尔基体糖基化缺陷。第一种方法是基于用[2-(3)H]甘露糖对培养的哺乳动物细胞进行直接代谢标记，然后对纯化的寡糖进行顺序提取和HPLC分析。第二种方法基于铜催化的叠氮化物-炔烃环加成（CuAAC）策略。我们建议使用炔烃标记的唾液酸（SialNAl）来观察高尔基体糖基化效率。然后，它们代谢掺入新合成的糖蛋白中可以化学选择性地与互补的叠氮化物功能化荧光团偶联，并通过共聚焦激光扫描显微镜进行观察。总之，我们在此详细描述了我们在内质网和高尔基体N-糖基化领域的技术诀窍。
