Structure and Function of the Circumsporozoite Protein

疟原虫孢子子从蚊子的中肠壁到哺乳动物的肝脏进行了一次非凡的旅行。阐明孢子子的主要表面蛋白环孢子子蛋白（CSP）在这一过程中的作用是本研究的总体目标。我们一直在研究CSP的蛋白水解过程，到目前为止，已经发现当孢子体接触肝细胞时，加工发生，并且是有效入侵所必需的。具体来说，我们的数据表明，CSP的氨基端掩盖了羧基端的细胞粘附结构域，蛋白质水解裂解导致氨基末端的去除和该细胞粘附结构域的暴露。总的来说，我们的数据表明CSP有两个功能域，每个功能域在孢子子的旅程中都有不同的作用。本提案的目标是继续我们对这一重要蛋白质的结构-功能研究，以确定每个CSP结构域在哺乳动物宿主中的作用，并确定负责CSP切割的蛋白酶。具体目的是：1)阐明在哺乳动物宿主真皮中仅表达裂解形式CSP的孢子子突变体的表型。使用定量PCR、转录谱分析和共聚焦显微镜，我们将确定n端缺失突变是否留在皮肤中并开始在那里发育。2)通过在n端残基中产生具有潜在肝素结合活性的突变体，并使用生化技术确定TSR结构域的结合伙伴，确定每个CSP结构域的结合特性。3)鉴定裂解CSP的蛋白酶，确定其在子孢子中的亚细胞位置。这些研究将使用条件诱变来删除候选蛋白酶基因，并使用抗血清来定位孢子体中的蛋白酶。总的来说，这些研究将阐明CSP如何在孢子子从蚊子到哺乳动物宿主的旅程中发挥作用，并为疟疾的控制和预防开辟新的途径。

Plasmodium sporozoites make a remarkable journey from the mosquito midgut wall to the mammalian liver. It is the overall goal of this proposal to elucidate the role of the sporozoite's major surface protein, the circumsporozoite protein (CSP) in this journey. We have been studying the proteolytic processing of CSP and thus far, have found that processing occurs when sporozoites contact hepatocytes and is required for efficient invasion. Specifically, our data indicate that the amino-terminus of CSP masks the cell-adhesive domain in the carboxy-terminus and that proteolytic cleavage results in removal of the amino-terminus and exposure of this cell-adhesive domain. Overall our data suggest that CSP has two functional domains, each with distinct role(s) during the sporozoite's journey. The goal of this proposal is to continue our structure-function work on this important protein to define the role of each CSP domain in the mammalian host and to identify the protease responsible for CSP cleavage. The specific aims are: 1) To elucidate the phenotype of sporozoite mutants which expressed only the cleaved form of CSP in the dermis of the mammalian host. Using quantitative PCR, transcriptional profiling and confocal microscopy, we will determine whether N-terminal deletion mutants remain in the skin and begin their development there. 2) To determine the binding properties of each CSP domain by generating mutants in N-terminal residues that we have identified as having potential heparin-binding activity and using biochemical techniques to determine the binding partner of the TSR domain. 3) To identify the protease that cleaves CSP and determine its subcellular location in the sporozoite. These studies will be performed using conditional mutagenesis to delete candidate protease genes and using antisera to localize the proteases in sporozoites. Overall these studies will elucidate how CSP functions in the sporozoite¿s journey from mosquito to mammalian host and should open up new avenues for the control and prevention of malaria.