Multivalent glycobiomaterials for specific recognition and binding by lectins
- Multivalente Glykobiomaterialien für die spezifische Erkennung und Bindung durch Lektine
Rosencrantz, Ruben R.; Elling, Lothar (Thesis advisor); Böker, Alexander (Thesis advisor)
Aachen : Publikationsserver der RWTH Aachen University (2015, 2016)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen, 2016
Glycans are one of the most complex biomolecules and are used in nature for various tasks from cell-cell adhesions and communication to invasion or pathogenic processes. The most important term in protein-glycan interaction is the "multivalent effect". This describes the boost in avidity as soon as the number of presented glycans in close proximity to each other is increased. In this work, we aimed for the design and evaluation of multivalent scaffolds based on polymers for the specific recognition by lectins. Two major approaches have been taken: 1) Glycopolymers on solid supports: With chemo-enzymatic synthesis routes we produced immobilized, highly multivalent glycopolymer brushes presenting various glycans based on N-acetyllactosamine (LacNAc) on surfaces like silicon or even gold. Glycosyltransferases β4GalT-1, β3GlcNAcT, and α3GalT were utilized in biocatalytic cascade reactions to build-up complex glycans on the surface. These surfaces were evaluated with various lectins from plants (GS-II, ECL, WGA) and human galectin-3 and proved to be outstanding in means of avidity combined with good anti-fouling properties. We designed biosensors covered with glycopolymer brushes. With electrochemical impedance spectroscopy (EIS) as well as localized surface plasmon resonance spectroscopy (LSPR) we followed the binding of lectins in up to two dimensions. Furthermore, we produced a gradient in brush length on silicon surfaces. Binding profiles for various lectins on different brush lengths were determined. This revealed interesting binding-profile-function relationships and was unique for each lectin. 2) Glycopolymers in solution: We developed block copolymers with a glycopolymer block based on hydroxyethylmethacrylate (HEMA) linked to N-acetylglucosamine (GlcNAc). The structures formed micelles in water and we proved strong binding of lectins (GS-II) to the sugar shell as well as the possibility of capturing hydrophobic dyes (Nile Red) within these nano-containers. Finally, the scavenging of bacterial enterotoxins with glyco-functionalized microgels was investigated within this work. Tri-saccharide known as Galili-structure (Galα1,3Galβ1,4GlcNAcβ-) was incorporated into the biocompatible polyethyleneoxide based gel and for the first time flow cytometry was used for determination of enterotoxin binding to the functionalized microgels. We evaluated gels loaded with LacNAc and Galili-structure and found strong binding of toxin TcdA receptor binding domain from Clostridium difficile to Galili-functionalized gels. In this interdisciplinary work, various multivalent scaffolds for specific lectin binding were designed, produced and evaluated with various analytical and bioanalytical methods. Possible applications range from deep and quantitative characterization of lectins to biomedical use and glyco-medicine. The complexity of glycans and their interactions with proteins force the combination of different disciplines from biotechnology, chemistry, to engineering and medicine to fulfill the needs of sophisticated novel sugar-based materials.