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Abstract
Bioorthogonal chemical reporters are novel reagents in the field of chemical glycobiology that can be used for in vivo imaging of glycoproteins in cultured cells. Unlike large, bulky genetically encoded tags such as green fluorescent protein (GFP), these reporters are small molecules that can be incorporated into the cells glycan biosynthetic machinery without harming the biological system. One such reporter, N-azidoacetylmannosamine (ManNAz), is a sugar analog that is capable of integrating into nascent glycoproteins as sialic acid residues. A chemical handle can then be covalently linked to the azido group of incorporated ManNAz residues, allowing for the visualization of the modified glycoproteins using fluorescent probes that recognize this handle. Our goal is to use this technique (known as click chemistry) to investigate the nature and dynamics of glycoprotein storage in lysosomal storage disorders such as mucolipidosis II (ML-II), a congenital disease characterized by improper catabolism and accumulation of undigested macromolecules. Employing both epifluorescence and confocal microscopy, we have successfully visualized the accumulation of sialic acid-containing glycoproteins in the lysosomes of ML-II cells. Furthermore, the click chemistry has allowed us to view the dynamics of extracellular matrix (ECM) glycoprotein synthesis and turnover within WT cells. The application of different azide sugar precursors in other lysosomal storage disease tissues should provide us with additional ways to study the trafficking and turnover of glycoproteins in these cells. Our findings could facilitate new methods for lysosomal disease diagnosis and will provide unprecedented opportunities to track glycoprotein dynamics and turnover in affected cell types.