Glycobiological Analysis of Plasmodium-Vector Host Interactions

疟原虫-载体宿主相互作用的糖生物学分析

• 批准号: 7531216
• 负责人: Rhoel David Ramos Dinglasan
• 金额: $ 16.2万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7531216
• 关键词: Affinity; Affinity Chromatography; Aminopeptidase; Anabolism; Antibodies; Antigen Targeting; Bacterial Adhesins; Binding; Cellular biology; Chondroitin Sulfates; Concanavalin A; Core Protein; Culicidae; Development; Disaccharides; Double-Stranded RNA; Enzymes; Epitopes; Gene Silencing; Glycoconjugates; Glycolipids; Glycoproteins; Glycosaminoglycans; Grant; Immune Sera; Label; Lectin; Ligands; Link; Liquid Chromatography; Malaria; Malaria Vaccines; Mass Spectrum Analysis; Mediating; Methodology; Midgut; Molecular; O-Glycans Biosynthesis Pathway; Oligosaccharides; Parasites; Peptides; Phenotype; Plasmodium; Plasmodium berghei; Plasmodium falciparum; Polysaccharides; Post-Translational Protein Processing; Process; Progress Reports; Proteins; Proteoglycan; Proteomics; RNA Interference; Recombinant Proteins; Recombinants; Research; Research Design; Research Personnel; Role; Transferase; Translations; Vaccines; Variant; Work; base; cellular microvillus; chondroitin sulfate glycosaminoglycan; glycosyltransferase; homologous recombination; in vivo; insight; knock-down; knockout gene; mutant; parasite invasion; polypeptide; programs; receptor; research study; sulfation; sulfotransferase; tandem mass spectrometry; transmission process; vector; xylosyltransferase

DESCRIPTION (provided by applicant): Malaria transmission entails development of the Plasmodium parasite in the mosquito. An understanding of not only protein-protein but protein-glycan interactions are needed before we can completely dissect the molecular mechanisms involved in Plasmodium ookinete invasion of the mosquito midgut. Objectives: This is a proposal to assess the role of protein-glycan interactions during Plasmodium ookinete invasion of the midgut. To do so, we aim to further identify and characterize mosquito midgut glycan ligands and the protein core(s) to which the glycans are attached. We will then identify the cognate lectin-like receptors on the ookinete and characterize their functional role in vivo. Research Design: The research plan includes: a) functional analyses of mosquito midgut glycoconjugates during Plasmodium invasion through RNAi knock-down of mosquito glycosyl- and sulfo-transferases, b) proteomic identification by mass spectrometry of mosquito coretin gene knockout lines). Cellular glycosyltransferases are involved in posttranslational and co-translation modification of proteins. As proof of principle, by RNAi, we were able to diminish enzymatic activity of the primary midgut glycosyltransferase involved in initiating glycosaminoglycan (GAG) biosynthesis on polypeptides. This resulted in >90% inhibition of parasite development in the mosquito. We also provide evidence for the use of lectin-affinity chromatography followed by tandem mass spectrometry to identify glycoproteins that are recognized by specific lectins. Conversely, we anticipate that the process will be successful in identifying lectins that can bind to defined glycan moieties. Lastly, we propose to produce lectin gene knockout lines and assess the midgut invasion phenotype in vivo. Summary: An effective malaria vaccine remains elusive. The characterization of these mosquito ligands and parasite receptors offer us additional target antigens toward the development of malaria transmission-blocking vaccines and provide us critical insight into parasite and midgut cell biology and vector host-parasite interactions.

描述（由申请人提供）：疟疾传播需要蚊子体内疟原虫的发育。了解不仅蛋白质，但蛋白质-聚糖的相互作用，我们才能完全解剖疟原虫动合子入侵蚊子中肠的分子机制。目的：这是一个建议，以评估在疟原虫动合子入侵中肠的蛋白质聚糖相互作用的作用。为此，我们的目标是进一步识别和表征蚊子中肠聚糖配体以及聚糖所连接的蛋白质核心。然后，我们将确定同源凝集素样受体的动合子和其功能的特点在体内的作用。研究设计：研究计划包括：a）通过RNAi敲低蚊子糖基转移酶和磺基转移酶，在疟原虫入侵期间对蚊子中肠糖缀合物进行功能分析，B）通过质谱法对蚊子核心蛋白基因敲除系进行蛋白质组学鉴定。细胞糖基转移酶参与蛋白质的翻译后和共翻译修饰。作为原理的证明，通过RNAi，我们能够降低参与启动多肽上的糖胺聚糖（GAG）生物合成的主要中肠糖基转移酶的酶活性。这导致蚊子中寄生虫发育的抑制>90%。我们还提供了证据，使用凝集素亲和层析，然后串联质谱法，以确定被特定凝集素识别的糖蛋白。相反，我们预计该方法将成功地鉴定出可以与确定的聚糖部分结合的凝集素。最后，我们建议生产凝集素基因敲除株系，并在体内评估中肠侵袭表型。摘要：一种有效的疟疾疫苗仍然难以捉摸。这些蚊子配体和寄生虫受体的特性为我们提供了额外的靶抗原疟疾传播阻断疫苗的发展，并为我们提供了重要的洞察寄生虫和中肠细胞生物学和载体宿主-寄生虫相互作用。