Identification of PhIL1 a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide
Identification of PhIL1 a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide
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DOI：
10.1128/ec.00114-06
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发表时间：
2006-10-01
期刊：
影响因子：
--
通讯作者：
Ward, Gary E.
中科院分区：
文献类型：
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作者：
Gilk, Stacey D.;Raviv, Yossef;Ward, Gary E.
弓形虫原虫的膜是一种独特的三层双层结构，由质膜和两层紧密相对的内膜复合体组成。膜的完整膜蛋白可能在宿主细胞的识别、附着和入侵中发挥关键作用，但很少有这样的蛋白被鉴定出来。这在很大程度上是因为寄生虫表面由一系列丰富的、高度免疫原性的糖基磷脂酰肌醇(GPI)锚定蛋白主导，这使得识别非GPI连接蛋白变得困难。为了识别这类蛋白质，我们开发了一种使用疏水性、可光激活的化合物5-[I-125]碘化萘-1-叠氮(INA)的放射性标记方法。INA可以被光敏的荧光染料激活；通过将这些荧光染料限制在膜上，[I-125]INA标记将选择性地针对膜上非GPI锚定的膜包埋蛋白。我们在这里证明了三种已知的膜蛋白确实可以通过INA的光敏化来标记。此外，这种方法还发现了一种新的22 kDa蛋白质，名为PhIL1(光敏INA标记蛋白1)，具有意想不到的特性。虽然PhIL1的INA标记与完整的膜蛋白一致，但该蛋白既没有跨膜结构域，也没有预测的脂质修饰部位。PhIL1在顶端复合体寄生虫中保守，定位于寄生虫的外围，集中在锥体底部的顶端。洗涤剂提取和免疫定位数据表明，PhIL1与寄生虫的细胞骨架有关。
The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[I-125] iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [I-125]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.