Faculty of Science, University of Zagreb, Zagreb, Croatia.
PLoS One. 2013;8(1):e54672. doi: 10.1371/journal.pone.0054672. Epub 2013 Jan 15.
Glycans are essential regulators of protein function and are now in the focus of research in many physiological and pathophysiological processes. There are numerous modes of regulating their biosynthesis, including epigenetic mechanisms implicated in the expression of glyco-genes. Since N-glycans located at the cell membrane define intercellular communication as well as a cellular response to a given environment, we developed a method to preferentially analyze this fraction of glycans. The method is based on incorporation of living cells into polyacrylamide gels, partial denaturation of membrane proteins with 3 M urea and subsequent release of N-glycans with PNGase F followed by HPLC analysis. Using this newly developed method, we revealed multiple effects of epigenetic inhibitors Trichostatin A, sodium butyrate and zebularine on the composition of N-glycans in human cells. The induced changes were found to be reversible after inhibitor removal. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, wherein surface glycans play an important role, the presented work contributes to our understanding of their efficiency in altering the N-glycan profile of cancer cells in culture.
糖链是蛋白质功能的重要调节剂，目前正成为许多生理和病理生理过程研究的焦点。有许多调节糖链生物合成的方式，包括参与糖基因表达的表观遗传机制。由于位于细胞膜上的 N-糖链定义了细胞间的通讯以及细胞对特定环境的反应，我们开发了一种优先分析糖链这一分支的方法。该方法基于将活细胞掺入聚丙烯酰胺凝胶中，用 3 M 尿素部分变性膜蛋白，然后用 PNGase F 释放 N-糖链，再进行 HPLC 分析。使用这种新开发的方法，我们揭示了表观遗传抑制剂 Trichostatin A、丁酸钠和 zebularine 对人细胞 N-糖链组成的多种影响。发现抑制作用消除后诱导的变化是可逆的。鉴于目前许多表观遗传抑制剂被探索作为癌症治疗的一种治疗策略，其中表面糖链起着重要作用，因此本工作有助于我们了解它们在改变培养的癌细胞 N-糖链谱方面的效率。
