Role of erythrocyte band 3 in malaria adhesion

恶性疟原虫感染引起疟疾，导致约435，000人死亡，其中大部分是儿童， 疟疾流行区每年。恶性疟原虫的一个明显特征是隔离;感染的粘附 含有成熟寄生虫的RBC（iRBC）对内皮细胞的作用。iRBC显示出称为旋钮的表面凸起 其介导iRBC与内皮的附着。寄生虫和宿主的蛋白质都位于结处 有助于细胞粘附表型。可以破坏旋钮相互作用的策略提供了潜在的 治疗手段减弱其粘合性能;然而，其精确机制仍然很差 明白利用噬菌体展示cDNA筛选，我们确定了一个新的结寄生虫蛋白PfGARP。P. 恶性疟原虫富含谷氨酸的蛋白质（PfGARP）被输出到RBC外部并结合到细胞外的 带3的段。PfGARP的这种新的定位表明在细胞粘附表型中的功能作用。 iRBC。基于这些发现，我们假设PfGARP有助于疟疾的细胞粘附 感染的人红细胞以PfEMP 1非依赖的方式通过一种新的 内皮表面的配体-受体复合物。我们将测试这个假设如下：具体目标 1：PfGARP与人红细胞/RBC相互作用的机制。实验支持来自 我们用已知的结标记PfEMP 1和KAHRP进行的免疫共定位研究，将PfGARP置于 旋钮由于与PfGARP结合的带3的截短形式也定位于旋钮，这提高了细胞的存活率。 PfGARP-带3复合物可能作为连接iRBC的PfEMP 1-独立相互作用发挥作用 到内皮细胞。我们建议确定带3和关键氨基酸的精确胞外段 介导带3-PfGARP相互作用的酸性残基。我们计划使用条带3的高亲和力肽， 评估PfGARP与人RBC结合的功能效应，包括粘附性、变形性和条带 3磷酸化。我们将采用免疫电镜，免疫沉淀和质谱法来研究 在旋钮上PfGARP复合物的定位和组成。将生成PfGARP敲除寄生虫菌株 通过多种细胞粘附试验评价iRBC的粘附特性。目标2：确定宿主 PfGARP的内皮受体。我们构建了人内皮细胞噬菌体展示cDNA文库 (TNFα-活化的HUVECs），并用重组PfGARP作为诱饵进行筛选。在几个潜在的受体中 在筛选中鉴定，我们目前正在表征一种单通道跨膜受体，称为 CLEC 14 A（C型凝集素结构域包含14 A）作为PfGARP的潜在结合配偶体。为了测试 PfGARP与CLEC 14 A结合以将iRBC募集到血管内皮，我们将使用最先进的 塔夫茨大学赫尔曼实验室为内皮细胞系开发的成像工具，用于监测实时动力学 的细胞粘附。总之，拟议的研究将有助于我们了解生物学 旋钮的功能和PfGARP在iRBC与微血管内皮细胞粘附中的作用。

Plasmodium falciparum infections cause malaria resulting in the deaths of ~435,000 people, mostly children, in malaria-endemic regions each year. A distinct feature of P. falciparum is sequestration; the adhesion of infected RBCs (iRBCs) containing mature parasites to endothelial cells. iRBCs display surface bumps known as knobs that mediate the attachment of iRBCs to endothelium. Both parasite and host proteins located at the knobs contribute to the cytoadhesion phenotype. Strategies that can disrupt interactions of knobs offer potential therapeutic means of attenuating their adhesive properties; however, their precise mechanisms remain poorly understood. Using phage display cDNA screens, we identified a new knob parasite protein termed PfGARP. P. falciparum glutamic acid-rich protein (PfGARP) is exported outside of the RBCs and binds to an extracellular segment of band 3. This novel localization of PfGARP suggests a functional role in the adhesion phenotype of iRBCs. Based on these findings, we hypothesize that PfGARP contributes to the cytoadhesion of malaria infected human RBCs to vascular endothelium in a PfEMP1-independent manner by engaging a novel ligand-receptor(s) complex on the endothelial surface. We will test this hypothesis as follows: Specific Aim 1: Mechanism of PfGARP interactions with human erythrocytes/RBCs. Experimental support comes from our immuno-colocalization studies with known knob markers, PfEMP1 and KAHRP, placing PfGARP at the knobs. Since a truncated form of band 3, which binds to PfGARP, is also localized to knobs, this raises the possibility that the PfGARP-Band 3 complex may function as an PfEMP1-independent interaction linking iRBCs to the endothelial cells. We propose to identify the precise ectoplasmic segment of band 3 and critical amino acid residues that mediate Band 3-PfGARP interaction. We plan to use the high-affinity peptides of band 3 for assessing the functional effects of PfGARP binding to human RBCs including adhesion, deformability, and band 3 phosphorylation. We will employ immuno-EM, immunoprecipitation and mass spectrometry to investigate the localization and composition of PfGARP complex on knobs. A PfGARP knockout parasite strain will be generated to evaluate the adhesive properties of iRBCs by multiple cytoadhesion assays. Aim 2: Identification of host endothelial receptors for PfGARP. We generated a phage display cDNA library from human endothelial cells (TNFα-activated HUVECs) and screened with recombinant PfGARP as bait. Among several potential receptors identified in the screens, we are currently characterizing a single-pass transmembrane receptor termed CLEC14A (C-type lectin domain containing 14A) as a potential binding partner of PfGARP. To test the possibility that PfGARP engages CLEC14A to recruit iRBCs to the vascular endothelium, we will use state-of-the-art imaging tools developed for endothelial cell lines in the Herman laboratory at Tufts to monitor real-time kinetics of cytoadherence ex vivo. Together, the proposed studies will contribute to our understanding of the biological function of knobs and role of PfGARP in the adhesion of iRBCs to microvascular endothelial cells.