SWEETSWAP · The Nuclear Frontier: Expanding the Horizons of Protein Glycosylation

Glycosylation is the most common post-translational modification (PTM), decorating two thirds of secreted human proteins. Often dysregulated in disease, the specific attached glycan influences folding and function. Extended glycosylation is supposed to exclusively occur in the secretory pathway. I propose that this model needs to be revised. My preliminary results—both computational and experimental—question this long-standing dogma and hint at a new kind of PTM: extended glycosylation in mammalian nuclei. I propose a research project that exhaustively determines the presence, biosynthesis, transport, and function of extended nuclear glycosylation as a new group of PTMs. This ambitious project will broaden the areas of cell biology that are modulated by glycosylation, launching many new inquiries into disease-relevant functions of glycans, such as gene expression regulation via glycosylation, beyond the known case of O-GlcNAc. I will measure and probe nuclear glycosylation with a broad array of computational and experimental methods, such as subcellular glycoproteomics, glycomics, glyco-CLIP, data mining, AI, genetic engineering, click chemistry, immunostaining, flow cytometry, and CRISPR/Cas9 knockout screens. My approach is structured into three main aims: (i) rigorously cataloguing which nuclear glycan structures are exposed on which proteins in which cell line/species, delineating a nuclear glycome, (ii) elucidating mechanistically how these molecules are biosynthesized and transported into the nucleus, and (iii) probing the functions of these novel PTMs in human cells. Each aim will further the central hypothesis of nuclear glycosylation and has planned experiments to address aspects from biosynthesis to function. Crucially, this research will resolve several curious observations from decades of glycobiology, leading to the rewriting of textbooks, and open up a new field of study with the function of glycans in nuclear processes.