Genetics and Biochemistry of Sialylation in Drosophila

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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.