Saeui Christopher T, Cho Kyung-Cho, Dharmarha Vrinda, Nairn Alison V, Galizzi Melina, Shah Sagar R, Gowda Prateek, Park Marian, Austin Melissa, Clarke Amelia, Cai Edward, Buettner Matthew J, Ariss Ryan, Moremen Kelley W, Zhang Hui, Yarema Kevin J
Department of Biomedical Engineering, Translational Tissue Engineering Center, The Johns Hopkins University, Baltimore, MD, United States.
Department of Pathology, The Johns Hopkins School of Medicine, Baltimore, MD, United States.
Front Chem. 2020 Feb 5;8:13. doi: 10.3389/fchem.2020.00013. eCollection 2020.
Sialylation, a post-translational modification that impacts the structure, activity, and longevity of glycoproteins has been thought to be controlled primarily by the expression of sialyltransferases (STs). In this report we explore the complementary impact of metabolic flux on sialylation using a glycoengineering approach. Specifically, we treated three human breast cell lines (MCF10A, T-47D, and MDA-MB-231) with 1,3,4-O-BuManNAc, a "high flux" metabolic precursor for the sialic acid biosynthetic pathway. We then analyzed N-glycan sialylation using solid phase extraction of glycopeptides (SPEG) mass spectrometry-based proteomics under conditions that selectively captured sialic acid-containing glycopeptides, referred to as "sialoglycosites." Gene ontology (GO) analysis showed that flux-based changes to sialylation were broadly distributed across classes of proteins in 1,3,4-O-BuManNAc-treated cells. Only three categories of proteins, however, were "highly responsive" to flux (defined as two or more sialylation changes of 10-fold or greater). Two of these categories were cell signaling and cell adhesion, which reflect well-known roles of sialic acid in oncogenesis. A third category-protein folding chaperones-was unexpected because little precedent exists for the role of glycosylation in the activity of these proteins. The highly flux-responsive proteins were all linked to cancer but sometimes as tumor suppressors, other times as proto-oncogenes, or sometimes both depending on sialylation status. A notable aspect of our analysis of metabolically glycoengineered breast cells was decreased sialylation of a subset of glycosites, which was unexpected because of the increased intracellular levels of sialometabolite "building blocks" in the 1,3,4-O-BuManNAc-treated cells. Sites of decreased sialylation were minor in the MCF10A (<25% of all glycosites) and T-47D (<15%) cells but dominated in the MDA-MB-231 line (~60%) suggesting that excess sialic acid could be detrimental in advanced cancer and cancer cells can evolve mechanisms to guard against hypersialylation. In summary, flux-driven changes to sialylation offer an intriguing and novel mechanism to switch between context-dependent pro- or anti-cancer activities of the several oncoproteins identified in this study. These findings illustrate how metabolic glycoengineering can uncover novel roles of sialic acid in oncogenesis.
唾液酸化是一种影响糖蛋白结构、活性和寿命的翻译后修饰，一直被认为主要受唾液酸转移酶（STs）表达的控制。在本报告中，我们使用糖工程方法探索了代谢通量对唾液酸化的互补影响。具体而言，我们用1,3,4-O-丁酰基甘露糖胺（一种唾液酸生物合成途径的“高通量”代谢前体）处理了三种人乳腺癌细胞系（MCF10A、T-47D和MDA-MB-231）。然后，我们在选择性捕获含唾液酸糖肽（称为“唾液酸化糖位点”）的条件下，使用基于糖肽固相萃取（SPEG）质谱的蛋白质组学分析N-聚糖唾液酸化。基因本体（GO）分析表明，在1,3,4-O-丁酰基甘露糖胺处理的细胞中，基于通量的唾液酸化变化广泛分布于各类蛋白质中。然而，只有三类蛋白质对通量“高度敏感”（定义为两个或更多唾液酸化变化达10倍或更大）。其中两类是细胞信号传导和细胞黏附，这反映了唾液酸在肿瘤发生中的众所周知作用。第三类——蛋白质折叠伴侣——出乎意料，因为糖基化在这些蛋白质活性中的作用几乎没有先例。高度通量响应的蛋白质都与癌症有关，但有时作为肿瘤抑制因子，有时作为原癌基因，有时则取决于唾液酸化状态，两者皆是。我们对代谢糖工程化乳腺癌细胞分析的一个显著方面是一部分糖位点的唾液酸化减少，这出乎意料，因为在1,3,4-O-丁酰基甘露糖胺处理的细胞中细胞内唾液酸代谢物“构建块”水平增加。唾液酸化减少的位点在MCF10A细胞（<所有糖位点的25%）和T-47D细胞（<15%）中较少，但在MDA-MB- c231细胞系中占主导（约60%），这表明过量唾液酸在晚期癌症中可能有害，癌细胞可以进化出防止过度唾液酸化的机制。总之，通量驱动的唾液酸化变化提供了一种有趣且新颖的机制，可在本研究中鉴定的几种癌蛋白的上下文依赖性促癌或抗癌活性之间切换。这些发现说明了代谢糖工程如何能揭示唾液酸在肿瘤发生中的新作用。
