Periodic Reporting for period 1 - Sialoglycan Array (Generation of a Cell-Based Sialoglycan Array to Decipher Biological Interactions and Functions of the Human Sialome)
Periodo di rendicontazione: 2018-09-01 al 2020-08-31
Every human cell is covered by a dense layer of glycans, chains of sugar molecules linked to each other in a combinatorial manner thereby forming short, long, linear, and branched structures. The biosynthesis of glycans takes place in the endoplasmic reticulum and Golgi system by more than 200 glycosyltransferase enzymes that build the diverse glycans that are attached to membrane or secreted glycoproteins and glycolipids. Glycans are frequently terminated by sialic acids that are a family of chemically diverse sugars that can be linked to underlying sugars via different chemical bonds. This process in carried out by 20 sialyltransferase isoenzymes with partially overlapping functions that are not fully elucidated. Due to this combinatorial diversity, human cells produce a vast repertoire of structurally diverse sialic acid-carrying glycans (sialoglycans) at the surface - the Sialome. The Sialome is involved in numerous molecular interactions at the cell surface and biological processes such as cell-cell interactions and cell-extracellular matrix interactions. The Sialome constitute the ligands for sialoglycan-binding proteins such as the Selectins and the Siglec immune receptor family that have regulatory functions in the immune system. Pathogens exploit the Sialome to infect host cells and aberrations in the Sialome have been associated with inflammation, autoimmunity, and cancer. For example, cancer cells are frequently found to present aberrant sialoglycans that are associated with tumor growth and progression. Dissection of the biological interactions with the Sialome could provide insights into these physiological and pathological processes providing novel opportunities for the development of therapeutic approaches targeting the Sialome in major diseases such as infection, autoimmunity, and cancer. So far, the field has mainly focused on developing analysis techniques for sialoglycans in biological samples and their chemical synthesis. However, research into the biological interactions and functions of the Sialome in health and disease is rather limited, because suitable methods to address the Sialome as well as specific sialoglycans in the natural context of the cell surface are lacking. The aim of this project was to develop a cell-based sialoglycan array for display of the Sialome in the natural glycan context of glycoconjugates and the cell membrane suitable for cell-based assays and dissection of the biological interactions with sialoglycan-binding proteins such as the Siglecs.
First, precise genome editing was used for rational combinatorial gene knock-out (CRISPR/Cas9) and knock-in (Zinc Finger Nuclease) of glycosyltransferase genes involved in the biosynthesis of sialoglycans in human embryonic kidney cells (HEK293) to produce a library of stable isogenic cells each lacking/displaying a specific feature of the Sialome. The cell library was validated genetically and the glycan phenotypes were confirmed with probes specific for sialoglycans. For example, by combinatorial knock-out and knock-in of the sialyltransferase isoenzymes a panel of isogenic cells was generated that allow now study of the specific functions of the 20 sialyltransferase isoenzymes. Second, the cell-based glycan array was used in high-throughput cell-based assays such as automated flow cytometry to dissect the binding specificity of sialoglycan-binding receptors in the natural glycan context of living cells. Novel insights into the binding specificity of the human immunomodulatory Siglec receptors and the underlying essential glycosyltransferase genes and biosynthetic steps were obtained. In particular, undescribed high selectivity of individual Siglecs for distinct sialoglycans was discovered. Third, using the cell-based glycan array resource, a panel of recombinant glycoproteins with customizable and homogenous sialoglycan decoration was produced. Binding studies using this panel revealed contribution of the protein backbone as determinant for highly specific recognition of sialoglycans for example by Siglecs.
The rational genetic engineering approach to display the human Sialome by isogenic cells fills the gap in the methodology to probe biological interactions with the entire Sialome as well as specific sialoglycans in the natural context of glycoconjugates and the cell surface. The library of isogenic cells that forms the cell-based sialoglycan is a self-renewable and stable resource biobanked at the host institute available for sharing upon request. The findings obtained in this project will advance the understanding of the biosynthetic steps involved in sialoglycan synthesis with particular focus on the specific functions of the sialyltransferase isoenzymes. Insights into the fine-binding specificities of glycan-binding proteins such as the Siglecs can advance the study into the role of these receptors in immunology for instance by providing detailed information about their sialoglycan ligands and essential enzymes for biosynthesis that can now be studied in the context of physiological and pathological processes related to these receptors such as inflammation and autoimmunity. Evidence for the involvement of specific protein sequences in sialoglycan recognition by Siglecs could prompt further investigation into this additional layer of information in glycan binding. The purified glycoproteins can further be explored for targeting of sialic acid-binding receptors in cellular assays and might proof useful for glycan-based targeting in immunology research. In conclusion, the generated cell-based sialoglycan array will advance research into the biological interactions with the human sialome with opportunity for discovery of principles in sialoglycan biosynthesis, fine-binding specificities of glycan-binding proteins and production of potentially therapeutic glycoproteins.