Structure and Function of the Circumsporozoite Protein

描述(申请人提供)：疟原虫子孢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。这项建议的总体目标是阐明子孢子的主要表面蛋白-环子孢子蛋白(CSP)在这一过程中的作用。我们一直在研究CSP的蛋白分解过程，到目前为止，我们已经发现，当子孢子接触肝细胞时，该过程发生，是有效入侵所必需的。具体地说，我们的数据表明，CSP的氨基末端掩盖了羧基末端的细胞粘附域，蛋白水解性切割导致氨基末端的去除和细胞粘附域的暴露。总体而言，我们的数据表明CSP有两个功能结构域，每个结构域在子孢子的旅程中都有不同的作用(S)。这项建议的目的是继续我们对这一重要蛋白质的结构和功能的研究，以确定每个CSP结构域在哺乳动物宿主中的作用，并确定负责切割CSP的蛋白酶。其具体目的是：1)阐明在哺乳动物宿主真皮中仅表达裂解形式CSP的子孢子突变体的表型。利用定量聚合酶链式反应、转录图谱和共聚焦显微镜，我们将确定N末端缺失突变是否保留在皮肤中并在那里开始发育。2)通过在N端残基上产生突变体，并利用生化技术确定TSR结构域的结合伙伴，确定每个CSP结构域的结合性质。3)鉴定裂解CSP的蛋白水解酶并确定其在子孢子中的亚细胞位置。这些研究将使用条件突变来删除候选的蛋白酶基因，并使用抗血清来定位子孢子中的蛋白酶。总体而言，这些研究将阐明CSP如何在子孢子虫从蚊子到哺乳动物宿主的过程中发挥作用，并应为控制和预防疟疾开辟新的途径。 公共卫生相关性：这些研究与控制和预防世界上最重要的传染病之一有关。疟疾继续对生活在世界热带和亚热带地区的儿童和成人造成重大影响，部分原因是对我们最好的抗疟疾药物产生抗药性，以及缺乏有效的疫苗。该提案中描述的实验可以为新的药物疗法提供基础，并改进现有的疟疾疫苗开发方法。

DESCRIPTION (provided by applicant): 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 determines 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. PUBLIC HEALTH RELEVANCE: These studies are of relevance to the control and prevention of one of the most important infectious diseases in the world. Malaria continues to impact significantly, children and adults living in tropical and subtropical parts of the world, in part due to drug resistance to our best anti-malarials and to the lack of an effective vaccine. The experiments described in this proposal could provide the basis for new drug therapies and improve upon existing approaches to malaria vaccine development.