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Dissection of Glycan Function by Engineered Tissue Models

Periodic Reporting for period 3 - GlycoSkin (Dissection of Glycan Function by Engineered Tissue Models)

Reporting period: 2021-03-01 to 2022-08-31

Glycans decorate most proteins, cover cell membranes, and represent one of the four building blocks of life, together with nucleic acids, lipids, and amino acids. Yet, our understanding of how glycans influence the life of cells and organisms is limited, and only few functions have been molecularly dissected. Glycans present a huge structural diversity with species and cell- type specificity that underlie specific biological functions. However, more than half a century of research has been severely hampered by the complexity and technical difficulties with analyzing glycans. While, the glycome (all glycans in a cell or organism) is a difficult entry point for discovery, the glycogenome (all genes involved in glycosylation) in contrast is a feasible entry point, because most of the genes controlling glycosylation are now known, and there are fewer technical barriers especially with the emergence of gene editing technologies. Our research group has pioneered the “glycogenome entry” to functional glycomics using gene editing to simplify glycosylation in cells, and extended this strategy to develop a next generation approach using organotypic tissue models in combination with mass spectrometry to decipher glycan functions. The tissue model has provided the first evidence that aberrant glycosylation in cancer directly induce oncogenic features, and that glycosylation is essential for viral propagation. In this proposal, I will use step-by-step genetic deconstruction of glycosylation capacities in organotypic tissue models for broad discovery and dissection of specific structure-function relationships driving normal epithelial formation, cancer transformation and interaction between the host and the microbiome.
We have successfully created and characterized the first library of tissue models knocked out for key glycogenes controlling the major classes of glycan structures decorating eth surface of human cells.
We have demonstrated distinct phenotypic changes in skin formation associated with global loss of the main types of glycoconjugates found in man, illustrating the potential for use of organotypic tissue models for dissection of specific functions of glycans. The findings include critical roles of glycosphingolipids for skin formation and barrier functions, N-glycans in specialized secretion processes and cell migration and wound healing, mucin-type O-glycans for cell-cell and cell-matrix adhesion, and specialized O-glycans for regulated terminal differentiation of human keratinocytes. By use of our isogenic cell expanded library of glycoengineered cells we have also initiated the dissection of the importance of the specific glycan structures for attachment, propagation, release, and spread of herpes virus and identified distinct roles for N- and O-linked glycans as well as glycosphingolipids and glycosaminoglycans for virus infection.
The engineered library of tissues and the dissection platform serve as a sustainable, ethical, and easily accessible resource compared to traditional animal studies, and we believe this marks a substantial leap applicable widely to any tissue model. The tissue models and identified phenotypic cues opens for further dissection, and the strategy is applicable to any type of organotypic tissues model, which will lay the foundation for further genetic dissection and identification of the specific structural features involved.
Overview of GlycoSkin program