Genetics and Biochemistry of Sialylation in Drosophila

果蝇唾液酸化的遗传学和生物化学

• 批准号: 7118183
• 负责人: VLADISLAV M PANIN
• 金额: $ 27万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118183
• 关键词: Drosophilidae; arthropod genetics; axon; biochemistry; cell cell interaction; developmental genetics; enzyme activity; gene expression; gene mutation; glycosylation; mass spectrometry; posttranslational modifications; protein localization; protein purification; proteomics; sialyltransferases; two dimensional gel electrophoresis

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.

描述（由申请人提供）：我们研究的长期目标是了解在调节动物发育过程中调节细胞相互作用中扮演糖基化的作用。哺乳动物的囊酰化已成为密集研究的重点，因为它参与了重要的生物学过程，例如病原体宿主相互作用以及免疫和神经系统的功能。囊酰化途径中的缺陷与多种病理有关，包括肿瘤转移，突触可塑性受损和神经肌肉疾病。同时，溶解度的复杂性以及对遗传方法的极限对阐明哺乳动物中溶解化的生物学功能施加了很大的困难。该项目是针对果蝇模型系统中溶酶化的分子和遗传机制的全面理解。该系统的优点基于其先进的遗传方法，关于有据可查的发育事件的丰富信息，完整的基因组序列和相对较低的遗传冗余。拟议的多学科研究旨在全面地表征果蝇苯乙烯转移酶基因在分子和遗传水平上的全面表征，并阐明了溶解度在果蝇发育中的作用。该项目的具体目的之一是全面表征乙烯基转移酶的生化活性。为此，将在细胞培养物中表达乙烯基转移酶蛋白，使用亲和色谱纯化，并根据其酶活性测定。另一个具体目的是详细研究在不同发育阶段，辅助转移酶（基因和蛋白质）的表达模式。为了准确绘制辅助转移酶的表达，将使用不同的分子标记物用于免疫染色和原位杂交分析。通过通过几种遗传技术（包括基因靶向和RNAI方法）获得的辅酶基因基因相关的表型的全面表征，将在分子，细胞和生物水平上分析乙烯基转移酶的功能。最后，在该项目的框架中，将使用基于蛋白质组学的方法来鉴定溶解度的体内分子靶标。这项拟议的研究将阐明溶糖化溶解度在果蝇中的分子机制和生物学作用，并应阐明包括人类在内的哺乳动物中溶解的生物学功能。