Japan Chemical Innovation Institute, Chiyoda-ku, Tokyo 101-0051, Japan.
Anal Biochem. 2010 Oct 1;405(1):103-8. doi: 10.1016/j.ab.2010.06.002. Epub 2010 Jun 4.
Saccharide primers, such as dodecyl beta-lactoside (Lac-C12), are unique artificial precursors of glycolipid synthesis. In culture media supplemented with saccharide primers, they are taken up by the cells in the culture media and glycosylated by cellular glycosyltransferases in the Golgi apparatus to form pseudo-glycolipids. In this study, we examine the effects of various xenobiotics on glycolipid synthesis by implementing a novel method to analyze pseudo-glycolipids, mainly gangliosides, produced by ONS-76 medulloblastoma cells in a culture medium containing various xenobiotics. The ganglioside group of pseudo-glycolipids was effectively purified by using strong anion-exchange cartridges. The production of pseudo-gangliosides was stimulated by N-(n-butyl)deoxygalactonojirimycin (NB-DGJ), but was inhibited by castanospermine, 2-deoxy-2-fluoro-d-galactose, tunicamycin, and brefeldin A. Because the cells in the culture medium are exposed to the saccharide primers and xenobiotics at the same time, and are secreted in the culture medium in their glycosylated form, our method can be used to effectively analyze the direct effects of xenobiotics on ganglioside synthesis.
糖脂引物，如十二烷基 β-内酰胺（Lac-C12），是糖脂合成的独特人工前体。在补充了糖脂引物的培养基中，它们被细胞摄取，并在高尔基体内由细胞糖基转移酶糖基化，形成假糖脂。在这项研究中，我们采用一种新方法分析含各种外源物的培养基中产生的假糖脂（主要是神经节苷脂），以研究各种外源物对糖脂合成的影响。通过使用强阴离子交换小柱可有效纯化假神经节苷脂的神经节苷脂组。N-（正丁基）去氧半乳糖基神经氨酸（NB-DGJ）刺激假神经节苷脂的产生，但Castanospermine、2-脱氧-2-氟-D-半乳糖、衣霉素和布雷菲德菌素 A 则抑制其产生。由于培养基中的细胞同时接触糖脂引物和外源物，并以其糖基化形式分泌到培养基中，因此我们的方法可有效地分析外源物对神经节苷脂合成的直接影响。
