BACKGROUND & AIMS:
:The cell surface glycoconjugates of trypanosomatid parasites are intimately involved in parasite survival, infectivity, and virulence in their insect vectors and mammalian hosts. Although there is a considerable body of work describing their structure, biosynthesis, and function, little is known about the sugar nucleotide pools that fuel their biosynthesis. In order to identify and quantify parasite sugar nucleotides, we developed an analytical method based on liquid chromatography-electrospray ionization-tandem mass spectrometry using multiple reaction monitoring. This method was applied to the bloodstream and procyclic forms of Trypanosoma brucei, the epimastigote form of T. cruzi, and the promastigote form of Leishmania major. Five sugar nucleotides, GDP-alpha-d-mannose, UDP-alpha-d-N-acetylglucosamine, UDP-alpha-d-glucose, UDP-alpha-galactopyranose, and GDP-beta-l-fucose, were common to all three species; one, UDP-alpha-d-galactofuranose, was common to T. cruzi and L. major; three, UDP-beta-l-rhamnopyranose, UDP-alpha-d-xylose, and UDP-alpha-d-glucuronic acid, were found only in T. cruzi; and one, GDP-alpha-d-arabinopyranose, was found only in L. major. The estimated demands for each monosaccharide suggest that sugar nucleotide pools are turned over at very different rates, from seconds to hours. The sugar nucleotide survey, together with a review of the literature, was used to define the routes to these important metabolites and to annotate relevant genes in the trypanosomatid genomes.
背景与目标:
: 锥虫寄生虫的细胞表面糖缀合物在其昆虫载体和哺乳动物宿主中与寄生虫的存活,感染性和毒力密切相关。尽管有大量的工作描述了它们的结构,生物合成和功能,但对促进其生物合成的糖核苷酸库知之甚少。为了鉴定和定量寄生虫糖核苷酸,我们开发了一种基于液相色谱-电喷雾电离-串联质谱的分析方法,该方法使用多反应监测。该方法适用于布鲁氏锥虫的血流和原环形式,克氏锥虫的表外形态和利什曼原虫的前形态。五种糖核苷酸,GDP-α-d-甘露糖,UDP-α-d-N-乙酰葡萄糖胺,UDP-α-d-葡萄糖,UDP-α-吡喃半乳糖和GDP-β-l-岩藻糖对所有三个物种都是共有的; 一,UDP-α-d-半乳糖呋喃糖,是克鲁氏乳杆菌和L. major常见的; 三个,UDP-β-l-鼠李吡喃糖,UDP-α-d-木糖和UDP-α-d-葡萄糖醛酸,仅在克鲁氏乳杆菌中发现; 一个,GDP-α-d-阿拉伯吡喃糖,仅在L. major中发现。对每种单糖的估计需求表明,糖核苷酸池的翻转速率从几秒钟到几小时不等。糖核苷酸调查以及文献综述被用来定义通往这些重要代谢物的途径,并注释锥虫属基因组中的相关基因。