Kinetic Tracking of Plasmodium falciparum Antigens on Infected Erythrocytes with a Novel Reporter of Protein Insertion and Surface Exposure.
Kinetic Tracking of Plasmodium falciparum Antigens on Infected Erythrocytes with a Novel Reporter of Protein Insertion and Surface Exposure.
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作者：
细胞内疟原虫向宿主细胞输出许多蛋白质，并将一些蛋白质插入红细胞膜，使其能够与外部环境相互作用。虽然静态技术已经确定了一些表面暴露的蛋白质，但其他候选蛋白还没有明确的定位和膜拓扑确定。此外，出口动力学和膜插入机制在很大程度上仍未被探索。我们介绍了插入和表面暴露报告（RISE），一种连续无损跟踪抗原暴露在感染细胞上的方法。RISE利用NanoLuc的一个小的11个氨基酸（aa） HiBit片段插入到靶蛋白中，通过高亲和力互补检测表面暴露从而产生发光。我们跟踪了CLAG3的输出和表面暴露，CLAG3是一种与营养摄取相关的寄生虫蛋白，在整个恶性疟原虫在人红细胞中的循环过程中。我们的方法揭示了贩运和地表暴露的关键决定因素。c端跨膜结构域的移除导致输出失败。出乎意料的是，暴露的报告基因大小的一定增加改善了发光信号，但其他变化消除了表面信号，这表明细胞外表位的大小和电荷都影响膜插入。含有多个HiBit表位的较大插入物在细胞间的显著差异表明CLAG3在宿主膜上插入的复杂调控。因此，对CLAG3表面暴露的定量、连续跟踪揭示了决定该蛋白在宿主红细胞膜上的运输和插入的多种因素。RISE试验将使研究来自不同细胞内病原体的表面抗原成为可能。
Intracellular malaria parasites export many proteins into their host cell, inserting several into the erythrocyte plasma membrane to enable interactions with their external environment. While static techniques have identified some surface-exposed proteins, other candidates have eluded definitive localization and membrane topology determination. Moreover, both export kinetics and the mechanisms of membrane insertion remain largely unexplored. We introduce Reporter of Insertion and Surface Exposure (RISE), a method for continuous nondestructive tracking of antigen exposure on infected cells. RISE utilizes a small 11-amino acid (aa) HiBit fragment of NanoLuc inserted into a target protein and detects surface exposure through high-affinity complementation to produce luminescence. We tracked the export and surface exposure of CLAG3, a parasite protein linked to nutrient uptake, throughout the Plasmodium falciparum cycle in human erythrocytes. Our approach revealed key determinants of trafficking and surface exposure. Removal of a C-terminal transmembrane domain aborted export. Unexpectedly, certain increases in the exposed reporter size improved the luminescence signal, but other changes abolished the surface signal, revealing that both size and charge of the extracellular epitope influence membrane insertion. Marked cell-to-cell variation with larger inserts containing multiple HiBit epitopes suggests complex regulation of CLAG3 insertion at the host membrane. Quantitative, continuous tracking of CLAG3 surface exposure thus reveals multiple factors that determine this protein’s trafficking and insertion at the host erythrocyte membrane. The RISE assay will enable study of surface antigens from divergent intracellular pathogens.
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