double daggerDipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Mol Cell Proteomics. 2010 Jul;9(7):1437-48. doi: 10.1074/mcp.M900479-MCP200. Epub 2010 Mar 22.
Despite over a century of study of malaria parasites, parts of the Plasmodium falciparum life cycle remain virtually unknown. One of these is the early gametocyte stage, a round shaped cell morphologically similar to an asexual trophozoite in which major cellular transformations ensure subsequent development of the elongated gametocyte. We developed a protocol to obtain for the first time highly purified preparations of early gametocytes using a transgenic line expressing a green fluorescent protein from the onset of gametocytogenesis. We determined the cellular proteome (1427 proteins) of this parasite stage by high accuracy tandem mass spectrometry and newly determined the proteomes of asexual trophozoites and mature gametocytes, identifying altogether 1090 previously undetected parasite proteins. Quantitative label-free comparative proteomics analysis determined enriched protein clusters for the three parasite developmental stages. Gene set enrichment analysis on the 251 proteins enriched in the early gametocyte proteome revealed that proteins putatively exported and involved in erythrocyte remodeling are the most overrepresented protein set in these stages. One-tenth of the early gametocyte-enriched proteome is constituted of putatively exported proteins, here named PfGEXPs (P. falciparum gametocyte-exported proteins). N-terminal processing and N-acetylation at a conserved leucine residue within the Plasmodium export element pentamotif were detected by mass spectrometry for three such proteins in the early but not in the mature gametocyte sample, further supporting a specific role in protein export in early gametocytogenesis. Previous reports and results of our experiments confirm that the three proteins are indeed exported in the erythrocyte cytoplasm. This work indicates that protein export profoundly marks early sexual differentiation in P. falciparum, probably contributing to host cell remodeling in this phase of the life cycle, and that gametocyte-enriched molecules are recruited to modulate this process in gametocytogenesis.
尽管对疟原虫进行了一个多世纪的研究，但疟原虫生命周期的某些部分仍然知之甚少。其中之一是早期配子体阶段，这是一种圆形细胞，形态上类似于无性滋养体，在这个阶段，主要的细胞转化确保了随后的长形配子体的发育。我们开发了一种使用从配子发生开始表达绿色荧光蛋白的转基因系首次获得高度纯化的早期配子体制剂的方案。我们通过高精度串联质谱法确定了这个寄生虫阶段的细胞蛋白质组（1427 种蛋白质），并新确定了无性滋养体和成熟配子体的蛋白质组，总共鉴定出 1090 种以前未检测到的寄生虫蛋白。定量无标签比较蛋白质组学分析确定了三个寄生虫发育阶段的丰富蛋白簇。对早期配子体蛋白质组中富集的 251 种蛋白质进行基因集富集分析表明，推测分泌并参与红细胞重塑的蛋白质在这些阶段中是最具代表性的蛋白质组。早期配子体富集蛋白质组的十分之一由推测分泌的蛋白质组成，在这里称为 PfGEXPs（疟原虫配子体分泌蛋白）。通过质谱法在三个早期但不是成熟配子体样本中检测到保守亮氨酸残基内的疟原虫出口元件 pentamotif 的 N-末端加工和 N-乙酰化，进一步支持在早期配子发生中蛋白质出口的特定作用。先前的报告和我们实验的结果证实，这三种蛋白质确实在红细胞细胞质中分泌。这项工作表明，蛋白质的分泌在疟原虫的早期性别分化中具有深远的影响，可能有助于生命周期中这一阶段的宿主细胞重塑，并且配子体富集分子被招募来调节配子发生中的这个过程。
