PEG-gebundene Oligosaccharide als biomolekulare Erkennungsstrukturen auf Grenzflächen

  • PEG-bound oligosaccharides as biomolecular recognition structures on surfaces

Sauerzapfe, Birgit; Elling, Lothar (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2008)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2008

Abstract

The targeting of cells to biomaterial surfaces can be achieved by functionalization of the interface with specified biomolecular recognition structures. The aim is the production of biohybrid systems with nano- and micro-structured surfaces to control the molecular mechanism of the cell. The cell surface is very "sweet" and is surrounded by a thick layer of sugars. These sugar chains are arranged like antennas which act as recognition structures and information carriers. One important glycan structure is poly-N-acetyllactosamine (poly-LacNAc) that fulfils the major function during inter- and intracellular communication events. This sugar structure has been identified as important ligand for galectin-mediated cell adhesion to extra-cellular matrix glycoproteins (laminin, fibronectin). Therefore poly-LacNAc shall be synthesized by chemo-enzymatic methods, coupled to specialized surfaces and characterized for its usage in the biomaterial research. The first step of the synthesis strategy comprised the chemical synthesis of beta-glycosides of GlcNAc to facilitate a controlled coupling onto chemically functionalized surfaces (cooperation with Prof. Dr. Kren, ASCR, Czech Republic). The best result could be reached with the product beta-D-GlcNAc-linker-NH2-tBoc which can be easily attached to amino-reactive surfaces. In a further step the glycoside was converted as acceptor substrate by human and bacterial beta-1,4-galactosyltransferases for the production of LacNAc in a biocatalytical one-pot-reaction (cooperation with Dr. W. W. Wakarchuk, NRC, Canada). The fusion protein His6-propeptide-beta-4GalT-1 turned out to be the most productive enzyme. It showed a preference for hydrophobic substrates by quantitative conversion of 5 mM acceptor substrate in 3 h. The enzymatic synthesis of poly-LacNAc structures was accomplished by the combination of His6-propeptide-beta-4GalT-1 with beta-3GlcNAcT from Helicobacter pylori. Kinetic data and conditions for an optimal conversion of the hydrophobic substrates were determined. Even more, the produced defined poly-LacNAc structures were used as acceptor substrates for further enzymatic modifications corresponding to nature or for a specific enzymatic labelling with UDP-Gal-biotin. The functionalised LacNAc and poly-LacNAc structures could be easily purified by solid phase extraction. A deprotection step yielded the amino-terminating sugar structures which were subsequently coupled to functionalised microtiter plates. The functionalisation of amino reactive microtiter plates was a suitable small scale test system. The detection of the immobilized structures could be done with commercially available lectins or with the recombinantly expressed fungal galectin His6CGL2. Even more, an enzymatic procedure could be established to label sugar structures in a specific and more sensitive way. Finally, the binding of extracellular glycoproteins (laminin, fibronectin) to the functionalized microtiter plates could be shown. After establishing the chemical and enzymatic synthesis of the poly-LacNAc structures, their immobilization onto surfaces and their detection work should be started with biomaterial surfaces. Therefore functionalization experiments were done with PEG-star surfaces in a close cooperation with Prof. Dr. Möller (DWI, RWTH Aachen). It could be shown that it was not only possible to immobilize the glycans onto the surface but also to be build up an artificial extracellular matrix via the galectin His6CGL2 with collagen IV, fibronectin and laminin. Finally, these biofunctionalized surfaces could be used for targeted cell adhesion by poly-LacNAc- and galectin-mediated binding of collagen IV.

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