PROJECT 3 Endocytosis and recycling of glycoconjugates are basic homeostatic mechanisms for controlling the turnover of many cell surface proteins, including signaling receptors, adhesion molecules, and membrane transporters. Many inherited human disorders affect the trafficking and turnover of glycoconjugates, leading to serious clinical manifestations. Impaired recycling and secondary storage of glycans is common in many of these diseases but the mechanisms that underlie this accumulation and the identity of the affected molecules are difficult to determine since few effective methods for targeted visualization and capture of glycoconjugates are currently available. The chemical reporter strategy is emerging as a versatile technology to visualize and capture glycoconjugates. While useful for tracking bulk glycoconjugate flux, the metabolic interconversion of glycosylation precursors from one form into another (as nucleotide sugars) and the broad dissemination of precursors into multiple glycan classes has limited the selectivity of metabolic labeling with modified precursors and hampered its ability to report changes in specific classes of glycoconjugates. Thus, there is an urgent need to develop new technologies for the selective labeling of specific glycoconjugate structures on intact cells. We propose to take advantage ofthe unprecedented availability of recombinant mammalian glycosyltransferases generated by the core of this program project and of our capacity to produce well-characterized, modified nucleotide sugars to selectively tag different classes of glycoconjugate glycans in disease and control cells. Our Selective Exo-Enzymatic Labeling strategy (SEEL) will provide us with the ability to track, capture, and identify subsets of cell surface glycoconjugates, revolutionizing the manner in which these molecules can be analyzed in the context of human disease. Once optimized, these SEEL tools will be employed to dissect the mechanisms that underlie altered glycoconjugate recycling and storage and identify the molecules whose cell surface localization or secretion are most affected. We will apply our innovative technological platform to investigate the contribution of glycan recycling/turnover mechanisms to the cellular pathophysiology of two human diseases characterized by altered glycoconjugate storage, Niemann-Pick disease type C (NPC) and mucopolysaccharidosis type IIIB (MPSIIIB), using cell types most relevant for the disease process (neural cells). SEEL-tagged glycoconjugates will also be employed to characterize the glycan and protein components of the glycoproteome of labeled cells, providing opportunities to further define glycosyltransferase specificity and to isolate sensitive molecules in disease-relevant cell types.
Impaired recycling and secondary storage of glycans is common in many inherited diseases but the mechanisms that underlie this accumulation and the identity ofthe affected molecules are difficult to determine since few effective methods for targeted visualization and capture of glycoconjugates are currently available. The proposed project will develop and apply technology that can track, capture, and identify subsets of cell surface glycoconjugates, revolutionizing the manner in which these molecules can be analyzed in the context of human disease.
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