The objectives of this project are: (1) the development of chemical tools for fundamental studies of O-linked glycosylation, and (2) the identification of glycoprotein cancer biomarkers. Mucin-type O-linked glycosylation is a posttranslational modification of membrane and secreted proteins in higher eukaryotes. Its functions are poorly understood, but altered mucin-type O-linked glycosylation has been correlated with cancer growth and metastasis. Chemical tools are needed both for perturbing and detecting O-linked glycosylation on proteins and cells. The first major goal of this renewal application is to develop small molecule inhibitors of mucin-type O-linked glycosylation by targeting the polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) and UDP-GlcNAc/GalNAc C4-epimerase (GALE), enzymes that are required for O-glycan biosynthesis. The inhibitors will be used to probe the importance of O-linked glycans in tumor growth and metastasis. The second goal is to develop a chemical approach for rapid profiling of changes in mucin-type O-linked glycosylation associated with cancer. The approach involves metabolic labeling of O-linked glycoproteins within living animals using an azido analog of N-acetylgalactosamine (termed GalNAz). The labeled glycoproteins from serum and tissue samples will be chemically tagged with phosphine probes via Staudinger ligation, permitting their detection and identification using proteomic methods. Comparison of labeled species from normal and tumor-bearing mice may reveal new serum biomarkers of disease. The final goal is to develop an analogous method for detection of protein O-fucosylation, a recently discovered form of O-linked glycosylation with mysterious functions. Cell-surface sugars are known to participate in many normal and disease processes, but have not yet been exploited as targets for drugs or clinical diagnostics. This research will advance our understanding of the roles cell-surface sugars play in tumor growth and metastasis. The chemical tools developed in this project may produce a new generation of anti-cancer drugs and new clinical tests for early diagnosis.
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