Synthesis of neo-glycoproteins for binding studies and scavenging of Clostridium difficile toxin A - of microgels and biosensors

  • Synthese von Neo-Glykoproteinen für Bindungsstudien und das Abfangen von Clostridium difficile Toxin A - von Mikrogelen und Biosensoren

Heine, Viktoria; Elling, Lothar (Thesis advisor); Schwaneberg, Ulrich (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Clostridium difficile infections cause enormous costs in the health care sector. The bacterium colonizes the human intestinal tract and secretes toxins that destroy the epithelial cell layer of the colon. The toxins bind to cell-surface glycans on human intestinal cells and induce processes that eventually lead to cell death. Since toxin A comprises a carbohydrate recognition domain with combined repetitive oligopeptide domains, it can bind multiple glycan ligands at once. This feature can be exploited for increasing the interaction strength between ligand and toxin by multivalency and can be transferred to other toxins comprising carbohydrate recognition domains. To find the best possible toxin ligand for C. difficile toxin A, we established an in-plate ligand library. Multivalent neo-glycoproteins served as scaffold for complex glycosylation patterns to assemble a variety of 40 glycan structures. Screening of this library with the receptor domain of toxin A presented Lewisy-Lewisx-decorated BSA as promising toxin scavenger. Tests with the holotoxin confirmed the findings and the ligand was produced for in vitro applications. For the production of the glycan (Lewisy-Lewisx), a set of efficient fucosyltransferases was established and tested with a range of new substrates (N-acetyllactosamine tetrasaccharides), revealing unexpected fucosylation patterns. The toxin A scavenger was produced and coupled to BSA. They were further utilized for coupling to microgels. Neo-glycoproteins and neo-glycoprotein-presenting microgels were able to protect human cells from toxin A. Furthermore, the neo-glycoproteins showed a good affinity to the Cholera toxin. Hence, Lewisy-Lewisx-BSA is considered a one-for-two scavenger. To enable a deeper understanding of the binding behavior, a neo-glycoprotein biosensor was established. BSA-neo-glycoproteins carrying N-acetyllactosamine were bound to the biosensor and glycosylated on-chip. Online measurements enabled the real-time investigation of kinetic binding processes between glycan and enzyme or glycan and lectin via electrochemical impedance spectroscopy for the first time. This approach could be transferred to a variety of interaction partners. Summarizing, this work paves the way towards the treatment of bacterial-associated diseases by establishing toxin scavengers and developing novel analytical methods for toxin-ligand interactions.