Glycobiological Analysis of Plasmodium-Vector Host Interactions

描述(申请人提供)：疟疾传播需要在蚊子中发展疟原虫寄生虫。我们不仅需要了解蛋白质-蛋白质之间的相互作用，而且还需要了解蛋白质-多糖之间的相互作用，然后才能完全剖析蚊虫中肠运动疟原虫入侵的分子机制。目的：这是一项评估蛋白质-糖链相互作用在动眼疟原虫侵入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鉴定蚊子中肠多糖的配体和附着在多糖上的蛋白质核心(S)。然后，我们将鉴定运动细胞上的同源凝集素样受体，并表征它们在体内的功能。研究设计：研究计划包括：a)通过RNAi敲除蚊子糖基转移酶和磺基转移酶，分析蚊子中肠糖结合物在疟原虫入侵过程中的功能；b)用质谱仪鉴定蚊子核心蛋白基因敲除株的蛋白质组。细胞糖基转移酶参与蛋白质的翻译后修饰和共翻译修饰。作为原理的证明，通过RNAi，我们能够降低启动多肽上糖胺聚糖(GAG)生物合成的主要中肠糖基转移酶的酶活性。这导致&gt；90%抑制蚊子体内寄生虫的发展。我们还提供了使用凝集素亲和层析和串联质谱仪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鉴定出能够与已定义的糖链结合的凝集素。最后，我们建议建立凝集素基因敲除系，并评估体内的中肠侵袭表型。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提供了更多的靶抗原来开发疟疾传播阻断疫苗，并为我们提供了对寄生虫和中肠细胞生物学以及媒介宿主-寄生虫相互作用的重要洞察力。

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.