Engineering and Functional Activities of Polysialic acid

Nature has chosen glycans (sugar structures) to form the communication front of each living cell. A likely reason for this decision is the enormous permutation potential of sugars, meaning that sugar structures have virtually unlimited capacity to create and transport information. Recent work has provided impressive evidence of the multiple functions of a specific sugar polymer called polysialic acid (polySia) in the prevention and treatment of human diseases. However, due to its complexity, polySia is difficult to manufacture which impedes deeper investigations into size-function-relationships. The overall goal of this application is to establish a system that efficiently generates polySia with controlled chain length and to investigate its exact biological functions. For this purpose, certain human and bacterial enzymes are specifically modified by molecular biological methods and then used for plant pathway engineering approaches aiming for a controlled synthesis of polySia structures. Functional studies include evaluation of anti-inflammatory effects and in vivo stability (e.g. serum half-life). The two applicants are leading experts in complementary fields, molecular biology, biotechnology and immunology. With this expertise we expect to create a synergistic effect that generates a unique competitive advantage, opening new avenues for the synthesis and understanding of complex sugar compounds.

Glycosylation is a biochemical process in which a carbohydrate (i.e. glycan) is covalently attached to molecules, affecting various biological functions such as protein folding or protein-protein interactions. Sialylation is a particularly complex glycan modification in mammals in which the sugar sialic acid is incorporated at the end of an oligosaccharide chain. Protein sialylation plays a critical role in cellular communication, immune regulation and disease processes. The project characterised several novel enzymes and proteins involved in the complex human sialic acid biosynthetic pathway. This knowledge was used to engineer targeted glycan structures on recombinant proteins produced in a plant-based expression system. Plants do not produce protein-bound glycan structures with sialic acid and are therefore an optimal production system for targeted de novo synthesis Genome editing combined with synthetic biology was used to optimise the expression of recombinant proteins and the production of defined homogeneous glycans for functional studies. A number of proteins of therapeutic interest, including different monoclonal antibody formats, were characterised to assess the effect of engineered glycosylation profiles on antigen binding, virus neutralisation potency and immune receptor interaction. The results highlighted the importance of targeted glycosylation for recombinant antibody function. A particular challenge was the coordinated expression of genes from distantly related species, such as bacteria or mammals, in plants. Extensive and creative modifications, such as those affecting subcellular localisation, have enabled the introduction of novel mammalian glycan biosynthetic pathways, such as polysialylation and KDNylation, into plants. Innovative purification methods for the production of high-purity glycan substances have been established. These high-quality substances are now being used to investigate biomedically interesting activities, such as anti-inflammatory effects. In summary, plants represent a sustainable and environmentally friendly resource for the production of sialic acid-based products. The results of this project could serve as a model for intensive cross-species engineering of organisms for the production of innovative biomedical products. The outcome of the project will lay the foundation for a number of follow-on projects aimed at unlocking the full potential of plant-produced glycoengineered products for various applications, including diagnostic tools and optimised therapeutics for the treatment of infections or human diseases.