Metabolomics data.

Trypanosomatids have been shown to possess an exclusive and finely regulated biosynthetic pathway for de novo synthesis of fatty acids (FAs) and particularly of polyunsaturated fatty acids (PUFAs). The key enzymes for the process of unsaturation are known as desaturases. In this work, we explored the biocatalytic activity of the putative Δ6-desaturase (Tb11.v5.0580) in the native organism T. brucei, whose expression level varies dramatically between life cycle stages. Utilising FA analysis via GC-MS, we were able to elucidate i) via genetic manipulation of the level of expression of Δ6-desaturases in both procyclic (PCF) and bloodstream (BSF) forms of T. brucei and ii) via supplementation of the media with various levels of FA sources, that docosahexaenoic acid (22:6) and/or docosapentaenoic acid (22:5) are the products, while arachidonic acid (20:4) and/or docosatetraenoic acid (22:4) are the substrates of this Δ6-desaturase. Surprisingly, we were able to observe, via lipidomic analysis with ESI-MS/MS, an increase in inositol-phosphoryl ceramide (IPC) in response to the overexpression of Δ6-desaturase in low-fat media in BSF. The formation of IPC is normally only observed in the stumpy and procyclic forms of T. brucei. Therefore, the expression levels of Δ6-desaturases, which increases between BSF, stumpy and PCF, might be involved in the cascade(s) of metabolic events that contributes to these remodelling of the lipid pools and ultimately morphological changes, which are key to the transition between these life-cycle stages. We were in fact able to show that the overexpression of Δ6-desaturase is indeed linked to the expression of protein associated with differentiation (PAD1) in stumpy, and of the upregulation of some proteins and metabolites which are normally upregulated in stumpy and PCF.

研究已证实，锥虫体（Trypanosomatids）拥有一套专属且调控精密的脂肪酸（Fatty Acids, FAs）从头合成途径，尤其针对多不饱和脂肪酸（Polyunsaturated Fatty Acids, PUFAs）的合成。该不饱和化过程的关键酶类被命名为去饱和酶（desaturases）。
本研究针对布氏锥虫（T. brucei）内推定的Δ6-去饱和酶（Δ6-desaturase，Tb11.v5.0580）的生物催化活性展开探究，该酶的表达水平在其生命周期的不同阶段存在显著差异。本研究借助气相色谱-质谱联用仪（Gas Chromatography-Mass Spectrometry, GC-MS）开展脂肪酸分析，通过两个维度阐明该Δ6-去饱和酶的催化特性：其一，对布氏锥虫前循环型（procyclic form, PCF）与血流型（bloodstream form, BSF）的Δ6-去饱和酶表达水平进行基因调控；其二，在培养基中添加不同浓度的脂肪酸底物。最终确认，二十二碳六烯酸（docosahexaenoic acid, 22:6）和/或二十二碳五烯酸（docosapentaenoic acid, 22:5）为该酶的催化产物，而花生四烯酸（arachidonic acid, 20:4）和/或二十二碳四烯酸（docosatetraenoic acid, 22:4）则为其催化底物。
令人意外的是，借助电喷雾串联质谱（Electrospray Ionization Tandem Mass Spectrometry, ESI-MS/MS）开展脂质组学分析时，我们观察到：在低脂肪培养基中对血流型布氏锥虫进行Δ6-去饱和酶过表达后，其肌醇磷酸神经酰胺（inositol-phosphoryl ceramide, IPC）的含量出现上调。通常而言，肌醇磷酸神经酰胺的合成仅在布氏锥虫的矮胖型与前循环型中被检测到。由此可见，Δ6-去饱和酶的表达水平（从血流型、矮胖型至前循环型依次升高）可能参与了一系列代谢级联反应，这些反应介导了脂质池的重塑与最终的形态学变化，而这正是锥虫不同生命周期阶段转换的关键。
事实上，我们已证实：Δ6-去饱和酶的过表达确实与矮胖型锥虫内分化相关蛋白1（Protein Associated with Differentiation 1, PAD1）的表达相关，同时也能上调通常在矮胖型与前循环型锥虫中表达上调的部分蛋白质与代谢物。