Wang Qian, Fujioka Hisashi, Nussenzweig Victor
Department of Pathology, Michael Heidelberger Division of Immunology, New York University School of Medicine, New York, NY 10016, USA.
Cell Microbiol. 2005 Nov;7(11):1616-26. doi: 10.1111/j.1462-5822.2005.00579.x.
The plasma membrane of Plasmodium sporozoites is uniformly covered by the glycosylphosphatidylinositol (GPI)-anchored circumsporozoite (CS) protein. Sporozoites form in the mosquito midgut through a budding process that occurs within a multinucleate oocyst underneath the basal lamina of the gut. Earlier genetic studies established that normal sporozoite development requires CS. Mutant parasites lacking CS [CS (-)] do not form sporozoites. Ultrastructural analysis of the oocysts from these parasites revealed that there is an early block in the cytokinesis that occurs within the multinucleate oocysts to generate individual sporozoites. Parasites that are hypomorphic for CS expression gave rise to sporozoites with abnormal morphology. These results proved that CS plays a direct role in the maturation of oocysts and in the normal budding of sporozoites. In this article, we examined if the membrane localization of CS via a GPI-anchor, is crucial for its function during sporozoite formation. We generated three mutants in Plasmodium berghei CS, CS-DeltaGPI, CS-TM1 and CS-TM2. In CS-DeltaGPI, we deleted the signal sequence required for the addition of a GPI-anchor to CS. The resulting protein was found only in the cytoplasm of the oocyst. In CS-TM1 and CS-TM2, the GPI-anchor addition sequence of CS was substituted by the transmembrane domain and truncated (to different degrees) cytoplasmic tail of Plasmodium thrombospondin-related anonymous protein (TRAP). The resulting CS protein was detected on the plasma membrane of the oocysts. The amount of CS in the mutants was similar to that of wild type. The sporozoite budding and development were abrogated in both CS-DeltaGPI and CS-TM mutants. The ultrastructure of the mutant oocysts was indistinguishable from that of the CS (-) parasites. Our results suggest that the GPI-anchor of the CS protein is required for sporogenesis.
疟原虫子孢子的质膜被糖基磷脂酰肌醇（GPI）锚定的环子孢子（CS）蛋白均匀覆盖。子孢子在蚊子中肠通过出芽过程形成，该过程发生在肠道基膜下方的多核卵囊中。早期的遗传学研究表明，正常的子孢子发育需要CS。缺乏CS的突变寄生虫[CS（-）]不能形成子孢子。对这些寄生虫卵囊的超微结构分析表明，在多核卵囊中发生的胞质分裂存在早期阻滞，从而无法产生单个子孢子。CS表达量低的寄生虫产生形态异常的子孢子。这些结果证明CS在卵囊成熟和子孢子的正常出芽中起直接作用。在本文中，我们研究了通过GPI锚定的CS的膜定位在子孢子形成过程中对其功能是否至关重要。我们在伯氏疟原虫CS中产生了三个突变体，CS-DeltaGPI、CS-TM1和CS-TM2。在CS-DeltaGPI中，我们删除了向CS添加GPI锚所需的信号序列。产生的蛋白质仅存在于卵囊的细胞质中。在CS-TM1和CS-TM2中，CS的GPI锚添加序列被血小板反应蛋白相关无名蛋白（TRAP）的跨膜结构域和截短（不同程度）的细胞质尾取代。在卵囊的质膜上检测到了产生的CS蛋白。突变体中CS的量与野生型相似。CS-DeltaGPI和CS-TM突变体中的子孢子出芽和发育均被消除。突变卵囊的超微结构与CS（-）寄生虫的超微结构无法区分。我们的结果表明，CS蛋白的GPI锚是孢子形成所必需的。
