Department of Biomedical Engineering and the Translational Tissue Engineering Center, The Johns Hopkins University, Baltimore, Maryland, United States of America.
Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States of America.
PLoS One. 2018 May 30;13(5):e0195812. doi: 10.1371/journal.pone.0195812. eCollection 2018.
In this report we use 'high-flux' tributanoyl-modified N-acetylmannosamine (ManNAc) analogs with natural N-acetyl as well as non-natural azido- and alkyne N-acyl groups (specifically, 1,3,4-O-Bu3ManNAc, 1,3,4-O-Bu3ManNAz, and 1,3,4-O-Bu3ManNAl respectively) to probe intracellular sialic acid metabolism in the near-normal MCF10A human breast cell line in comparison with earlier stage T-47D and more advanced stage MDA-MB-231 breast cancer lines. An integrated view of sialic acid metabolism was gained by measuring intracellular sialic acid production in tandem with transcriptional profiling of genes linked to sialic acid metabolism. The transcriptional profiling showed several differences between the three lines in the absence of ManNAc analog supplementation that helps explain the different sialoglycan profiles naturally associated with cancer. Only minor changes in mRNA transcript levels occurred upon exposure to the compounds confirming that metabolic flux alone can be a key determinant of sialoglycoconjugate display in breast cancer cells; this result complements the well-established role of genetic control (e.g., the transcription of STs) of sialylation abnormalities ubiquitously associated with cancer. A notable result was that the different cell lines produced significantly different levels of sialic acid upon exogenous ManNAc supplementation, indicating that feedback inhibition of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE)-generally regarded as the 'gatekeeper' enzyme for titering flux into sialic acid biosynthesis-is not the only regulatory mechanism that limits production of this sugar. A notable aspect of our metabolic glycoengineering approach is its ability to discriminate cell subtype based on intracellular metabolism by illuminating otherwise hidden cell type-specific features. We believe that this strategy combined with multi-dimensional analysis of sialic acid metabolism will ultimately provide novel insights into breast cancer subtypes and provide a foundation for new methods of diagnosis.
在本报告中，我们使用天然 N-乙酰基以及非天然叠氮基和炔基 N-酰基的高通量三丁酰基修饰 N-乙酰甘露糖胺（ManNAc）类似物（具体为 1,3,4-O-Bu3ManNAc、1,3,4-O-Bu3ManNAz 和 1,3,4-O-Bu3ManNAl）来研究近正常 MCF10A 人乳腺细胞系中的细胞内唾液酸代谢，与早期 T-47D 和更晚期 MDA-MB-231 乳腺癌细胞系进行比较。通过测量细胞内唾液酸的产生与与唾液酸代谢相关基因的转录谱的联合分析，获得了唾液酸代谢的综合视图。转录谱显示，在没有 ManNAc 类似物补充的情况下，三种细胞系之间存在一些差异，这有助于解释与癌症相关的天然唾液酸糖蛋白谱的不同。仅在暴露于化合物时，mRNA 转录本水平发生了较小的变化，这证实了代谢通量本身可以是乳腺癌细胞中唾液酸糖缀合物显示的关键决定因素；这一结果补充了普遍与癌症相关的唾液酸化异常的遗传控制（例如 STs 的转录）的既定作用。一个显著的结果是，不同的细胞系在外源性 ManNAc 补充后产生了明显不同水平的唾液酸，这表明 UDP-GlcNAc 2-差向异构酶/ManNAc 激酶（GNE）的反馈抑制-通常被认为是调节唾液酸生物合成通量的“守门员”酶-不是限制该糖产生的唯一调节机制。我们代谢糖工程方法的一个显著特点是，它能够通过揭示否则隐藏的细胞类型特异性特征，根据细胞内代谢来区分细胞亚型。我们相信，这种策略与唾液酸代谢的多维分析相结合，将最终为乳腺癌亚型提供新的见解，并为新的诊断方法提供基础。
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