ERYTHROCYTE ADHESION IN FALCIPARUM MALARIA

恶性疟疾中的红细胞粘附

• 批准号: 6349870
• 负责人: Irwin W Sherman
• 金额: $ 25.12万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349870
• 关键词: Plasmodium falciparum; SDS polyacrylamide gel electrophoresis; affinity chromatography; antibody titering; antigen antibody reaction; cell adhesion; clinical research; conformation; electron microscopy; erythrocyte membrane; erythrocytes; freeze etching; high performance liquid chromatography; human subject; immunoprecipitation; intracellular parasitism; malaria; matrix assisted laser desorption ionization; membrane proteins; protein binding; protein structure function; proteolysis; vascular endothelium; western blottings

Plasmodium falciparum contributes to 1.5-2.7 million deaths annually. Cerebral malaria, a complication of P. falciparum infections, is associated with sequestration of erythrocytes in the blood vessels of the brain and is due to the adhesion of malaria-infected erythrocytes (PE) to the endothelial cells (EC) lining the microcirculation. Sequestration of PE in the placenta of pregnant women leads to complications that include low fetal birth weight and an increased risk of death. At present none of the therapies for malaria specifically target the adhesive phenomena associated with sequestration. Describing the molecular architecture of the PE and EC surfaces is critical to understanding the mechanisms that lead to sequestration and for developing anti-adhesive treatments. It is known that increased adhesiveness of the PE is due to parasite-induced changes in the erythrocyte membrane protein, band 3, as well as the surface exposure of parasite-encoded proteins. Though the amino acid sequences of band 3 critical to adhesion have been identified it is not known how this adhesin is formed. To define the possible role of band 3 aggregation and/or proteolysis in inducing conformational change, the lateral distribution of band 3 within the bilayer will be evaluated by freeze fracture microscopy and coordinated with electron micrographic immunolocalization of adhesive sites, as well as by the use of fluorescence photobleaching recovery, polarized fluorescence depletion and luminescence quenching. In addition, to biochemically characterize the structural changes in band 3 that could influence the disposition of adhesive sequences, attempts will be made to isolate truncated forms of band 3 and cleavage sites identified. Immunoprecipitation, immunoblotting as well as competitive inhibition of antibody binding will be used to identify common adhesive motifs in the band 3-related and the parasite-encoded adhesin Plasmodium falciparum erythrocyte membrane protein-1; estimates of their contribution to cytoadherence will be determined. Other PE adhesins as well as EC ligands of microvessels in the human brain, dermis and bone marrow that bind PE will be identified and characterized through the use of monoclonal antibodies, phage display and synthetic combinatorial library technologies, and peptide affinity chromatography. Once identified, it should be possible to design drugs (i.e. peptidomimetics, small molecules, antibodies) that could inhibit or reverse sequestration thus allowing for better and safer therapeutic modalities for individuals suffering from falciparum malaria.

恶性疟原虫每年造成150万至270万人死亡。脑疟疾是恶性疟原虫感染的一种并发症，与脑血管中红细胞的隔离有关，这是由于受疟疾感染的红细胞（PE）粘附在微循环的内皮细胞（EC）上。妊娠妇女胎盘中PE的隔离导致并发症，包括胎儿出生体重低和死亡风险增加。目前，没有一种疟疾治疗方法专门针对与隔离相关的粘附现象。描述PE和EC表面的分子结构对于理解导致隔离的机制和开发抗粘接处理至关重要。众所周知，PE的黏附性增加是由于寄生虫诱导的红细胞膜蛋白第3带的变化，以及寄生虫编码蛋白的表面暴露。虽然已经确定了对粘附至关重要的带3的氨基酸序列，但尚不清楚这种粘附素是如何形成的。为了确定带3聚集和/或蛋白质水解在诱导构象变化中的可能作用，将通过冷冻断裂显微镜和粘附位点的电子显微免疫定位，以及使用荧光光漂白恢复，偏振荧光耗尽和发光猝灭来评估双分子层内带3的横向分布。此外，为了生物化学表征可能影响粘附序列配置的第3带的结构变化，将尝试分离第3带的截断形式和确定的切割位点。免疫沉淀、免疫印迹以及抗体结合的竞争性抑制将用于鉴定带3相关和寄生虫编码的黏附素恶性疟原虫红细胞膜蛋白-1中的常见黏附基序；将确定它们对细胞粘附性的贡献。通过使用单克隆抗体、噬菌体展示和合成组合文库技术以及肽亲和层析，将鉴定和表征人脑、真皮和骨髓微血管中与PE结合的其他PE粘附素以及EC配体。一旦确定，就有可能设计出能够抑制或逆转隔离的药物（即拟肽剂、小分子、抗体），从而为恶性疟疾患者提供更好和更安全的治疗方式。