Glycobiological Analysis of Plasmodium-Vector Host Interactions

说明（由申请人提供）：疟疾传播需要蚊子体内疟原虫的发展。在我们完全剖析卵动体疟原虫入侵蚊子中肠的分子机制之前，不仅需要了解蛋白质之间的相互作用，还需要了解蛋白质与聚糖之间的相互作用。目的：这是一项评估蛋白-聚糖相互作用在卵胞体疟原虫侵袭中肠过程中的作用的建议。为此，我们的目标是进一步鉴定和表征蚊子中肠聚糖配体和聚糖附着的蛋白质核心。然后，我们将在卵胞体上鉴定同源的凝集素样受体，并表征它们在体内的功能作用。研究设计：研究计划包括：a)通过RNAi敲除蚊子糖基转移酶和亚砜转移酶对疟原虫入侵过程中蚊子中肠糖缀合物的功能分析；b)通过质谱法对蚊子核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修饰和共翻译修饰。作为原理证明，通过RNAi，我们能够降低初级中肠糖基转移酶的酶活性，该酶参与在多肽上启动糖胺聚糖（GAG）的生物合成。这导致蚊子体内的寄生虫发育被抑制了约90%。我们还提供了使用凝集素亲和层析和串联质谱来鉴定特定凝集素识别的糖蛋白的证据。相反，我们预计这一过程将成功地识别凝集素，可以结合到定义的聚糖部分。最后，我们建议建立凝集素基因敲除系，并在体内评估中肠入侵表型。摘要：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征为我们开发疟疾传播阻断疫苗提供了额外的靶抗原，并为我们了解寄生虫和中肠细胞生物学以及媒介宿主-寄生虫相互作用提供了重要的见解。

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.