This proposal describes a new technology that uses metabolically incorporated photocrosslinking sugars to discover and characterize binding interactions of sialic acid- modified glycoproteins and glycolipids. This technology relies on the synthetic preparation of photocrosslinking analogs of sialic acid or of its precursor, N-acetylmannosamine (ManNAc). Analogs are added to cultured cells, which metabolize the compounds, resulting in incorporation of photocrosslinkers into cellular sialosides. Upon UV irradiation, photocrosslinkers are activated and form a covalent crosslink with neighboring molecules. Western blotting or mass spectrometry is used to identify components of crosslinked complexes.
In Aim 1, we will develop the best photocrosslinking reagents by synthesizing a panel of analogs, varying the position of attachment of the photocrosslinker, the spacing between the photocrosslinker and the pyranose ring, and the identity of the photocrosslinker. We will measure the metabolism of these molecules to cellular sialosides, evaluate their ability to crosslink glycan-mediated interactions, and use shotgun proteomics to identify the proteins present in a crosslinked complex.
In Aim 2, we will gain information about the sialosides into which sugar analogs are incorporated by determine the analog tolerance of individual sialyltransferases. This knowledge will aid in the interpretation of crosslinking results and enable us to design analogs for specific applications.
In Aim 3, we will expand a sialyltransferase's tolerance for unnatural donor sugars, thereby making the first step toward specific functionalization of a defined subset of the sialome. Sialic acid-mediated interactions play essential roles in pathogen recognition, cell differentiation and adhesion, neural development, and cancer biology. This method will be immediately applicable to problems such as identifying cellular receptors for sialic acid-recognizing viruses like influenza or defining functional ligands for components of the innate immune system, such as Siglec-10. My group has the essential expertise in unnatural carbohydrate synthesis, mammalian cell culture, assay development, and yeast engineering to carry out the proposed experiments. We will rely on advice and instrumentation of my colleague, Dr. Steven Patrie, for mass spectrometry experiments.
Carbohydrates cover the surfaces of all eukaryotic cells and determine how they interact with other cells, with foreign pathogens, and with molecules in the environment. Despite their critical roles, carbohydrate interactions are fleeting and therefore difficult to study. I propose the use of unnatural carbohydrates that, when activated by light, permanently bind to their neighbors, providing a "snapshot" of carbohydrate interactions.
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