Genetics and Biochemistry of Sialylation in Drosophila

描述(申请人提供)：我们研究的长期目标是了解在动物发育过程中糖基化在调节细胞相互作用中的作用。哺乳动物唾液酸化由于参与重要的生物学过程，如病原体与宿主的相互作用以及免疫和神经系统的功能，已成为深入研究的焦点。唾液酸化途径的缺陷与多种病理过程有关，包括肿瘤转移、突触可塑性受损和神经肌肉疾病。同时，唾液酸化的复杂性和遗传方法的局限性给阐明哺乳动物唾液酸化的生物学功能带来了极大的困难。本项目旨在全面了解果蝇模型系统唾液酸化的分子和遗传机制。该系统的优势在于其先进的遗传方法、关于发育事件的充分记录的丰富信息、完整的基因组序列以及相对较低的遗传冗余。这项多学科研究的目的是在分子和遗传水平上对果蝇唾液酸基转移酶基因进行全面的表征，并阐明唾液酸化在果蝇发育中的作用。本项目的具体目标之一是全面表征唾液酸基转移酶的生化活性。为此，唾液酸基转移酶蛋白将在细胞培养中表达，用亲和层析纯化，并测定其酶活性。另一个特定的目的是详细研究唾液酸基转移酶(基因和蛋白质)在不同发育阶段的表达模式。为了准确定位唾液酸基转移酶的表达，将使用不同的分子标记进行免疫染色和原位杂交分析。唾液酸基转移酶的功能将通过几种遗传技术，包括基因打靶和RNAi方法获得的唾液酸转移酶基因相关表型的综合表征，在分子、细胞和生物水平上进行分析。最后，在该项目的框架内，将使用基于蛋白质组学的方法来确定唾液酸化的体内分子靶点。这项拟议的研究将阐明唾液酸化在果蝇中的分子机制和生物学作用，并将有助于阐明唾液酸化在包括人类在内的哺乳动物中的生物学功能。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the role that plays glycosylation in regulating cell interactions during animal development. Mammalian sialylation has become the focus of intensive investigation because of its involvement in important biological processes, such as pathogen-host interactions and the functioning of the immune and nervous systems. Defects in the sialylation pathway have been implicated in multiple pathologies, including tumor metastases, impaired synaptic plasticity, and neuromuscular disorders. At the same time, the complexity of sialylation, and the limit on genetic approaches impose significant difficulties on elucidating biological functions of sialylation in mammals. This project is directed towards a comprehensive understanding of molecular and genetic mechanisms of sialylation in the Drosophila model system. The advantages of this system are based on its advanced genetic approaches, abundance of information on well-documented developmental events, the complete genome sequence, and relatively low genetic redundancy. The proposed multidisciplinary research is aimed at a comprehensive characterization of the Drosophila sialyltransferase gene at the molecular and genetic levels, as well as elucidating the role of sialylation in Drosophila development. One of the specific aims of this project is to comprehensively characterize the sialyltransferase biochemical activity. To this end, the sialyltransferase protein will be expressed in cell culture, purified using affinity chromatography, and assayed for its enzymatic activity. Another specific aim is to investigate in detail the expression pattern of the sialyltransferase (both gene and protein) during different developmental stages. To precisely map the expression of the sialyltransferase, different molecular markers will be used in immunostaining and in situ hybridization analyses. The function of the sialyltransferase will be analyzed at molecular, cellular and organismal levels by comprehensive characterization of the sialyltransferase gene-associated phenotypes obtained by several genetic techniques, including gene targeting and RNAi approaches. Finally, in the framework of this project, the in vivo molecular targets of sialylation will be identified using a proteomics- based approach. This proposed research will elucidate the molecular mechanism and biological role of sialylation in Drosophila and should shed light on biological functions of sialylation in mammals, including humans.