PROJECT SUMMARY/ABSTRACT Title: A Metabolic Engineering Strategy to Map Sialyltransferase Glycosites Glycoprotein sialylation plays an important role in a wide range of physiological and disease-related processes in areas such as viral infection, B cell development, and cancer metastasis. This posttranslational modification occurs primarily to terminate a growing glycan, and can modulate cell-cell signaling relevant to immune activation, receptor localization, as well as bulk physical properties of the cell surface. Sialyltransferases, the family of enzymes responsible for installing this glycan-terminating sialic acid unit, have garnered significant attention recently, due in part to their frequent dysregulation in multiple cancer subtypes. More specifically, identifying the protein targets of a given sialyltransferase has led to increased understanding of the molecular link between enzymatic activity and tumor progression. Unfortunately, this is a challenging task, due to the fact that sialylation glycosites are not predictable based solely on primary protein sequence, but instead are defined by a combination of three-dimensional structure and current glycosylation state. For these reasons, the development of a tool for interrogating sialyltransferase glycosites would provide further insight into the molecular basis of sialylation in driving disease and normal physiological function. The sialyltransferase ST6Gal I (β-galactoside α-2,6-sialyltransferase I) catalyzes the formation of α-2,6- linkages between a glycan-terminating galactose unit and N-acetylneuraminic acid, and is implicated in multiple mechanisms for cancer progression. Additionally, this enzyme has recently attracted renewed attention based on the role its soluble, circulating form may have in extracellular sialylation. This latter function is proposed to be important to modulating inflammation. The current proposal seeks to develop a metabolic reporter system to identify sialyltransferase glycosites through bump/hole enzyme and substrate engineering. This approach will be applied to studying the protein targets of ST6Gal I, but attention will be paid to ensuring the strategy can be generalized to the remaining members of the sialyltransferase enzyme family. After in vitro optimization of a small molecule/enzyme pair, mass spectrometry-based glycoproteomic analysis will be used to identify ST6Gal I glycosites in transfected cells. Finally, a mouse model of airway inflammation will be used to probe the extracellular glycosites of ST6Gal I in an in vivo setting.