Synthetic Glycobiology (SynGlycTis)

Great advances have been made in the development of proto-cells based on giant unilamellar vesicles (GUVs). However, one essential functional element of all living cells still to be incorporated into such systems is a glycocalyx. This coating of complex carbohydrates extends up to 100 nm from the cell membrane and provides an adhesive layer that mediates interactions between different cell types, viruses and signalling molecules. In most cases, these interactions involve specific carbohydrate-binding proteins (lectins) which may be either soluble or membrane-bound. For example, fertilisation is initiated by a specific carbohydrate on the surface of the egg adhering to a specific lectin on the head of the sperm. Protein-carbohydrate interactions also mediate the endocytosis of many bacteria, viruses and bacterial toxins which stick to specific glycolipids on the cell membrane. Protein-carbohydrate interactions thus present a general strategy for enabling cell adhesion and cell entry. In this application we propose to design and create a modular toolbox of synthetic glycocalyx components and engineered lectins that will be attached to lipid membranes to enable reversible proto-cell adhesion and incorporated into virus-like particles to mediate proto-cell entry. The methodology will be exemplified through the construction of proto-cells that contain "proto-organelles" and the assembly and remodelling of "proto-tissues" in which multiple types of proto-cells are brought together in a pre-defined fashion to create more complex systems. SynGlycTis is a transnational application involving partners form the UK, France, Denmark, Germany and Austria. The Austrian subproject focusses on the engineering of lectins with non-canonical amino acids. The amino acid analogues can furnish proteins with chemistries beyond those of the 20 canonical amino acids prescribed by the genetic code. We plan to replace lectin binding site residues with non-canonical analogues in order to manipulate the carbohydrate-binding of the lectins. The synthetic lectins will be biochemically characterized and promising candidates subjected to 3D structure elucidation. Employing non-canonical amino acid chemistry we intend to perform bio-orthogonal conjugation reactions with small molecules on lectins functionalised with reactive non- canonical amino acids. The conjugated lectins will be used to decorate lipid membranes and virus-like particles for "proto-organelle" and "proto-tissue" formation.

In the collaborative ERASynBio project SynGlycTis, lipid doplets, so-called proto-cells, were successfully functionalized to promote their crosslinking, which led to the formation of synthetic proto-tissues for the first time. The surface of every living cell is covered in a forest of carbohydrates. Interactions between this sugar coating with proteins on the surface of other cells allows them to adhere to one another. Similarly, protein-sugar interactions at the cell surface allow viruses enter cells. In this project our aim was to create a toolbox of carbohydrate and protein components that allow natural adhesion and cell entry processes to be re-created in artificial proto-cells. We devised sugar-binding proteins into which specific chemical modifications were genetically introduced to tune their interaction with the sugar. To enhance the sugar binding, they were fused with each other to form complex superstructures. We coupled them with bacterial toxins and anchor groups, such as lipids or peptides, to facilitate their installation on proto-cells. Proto-cells decorated with the engineered sugar-binding proteins could be crosslinked and fused to form complex synthetic proto-tissues. The individual partners from Great Britain, France, Denmark, Germany and Austria in the SynGlycTis consortium combined their complementary expertise to systematically tackle the aims of the project. The Austrian subproject focused on the genetic encoding of the chemical modifications of the sugar-binding proteins and contributed in chemical protein ligation. The synthetic tissues resulting from SynGlycTis will find use in many applications from tissue engineering to industrial biotechnology.