Poly-N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures and subsequent binding of ECM glycoproteins. First, we synthesized beta-GlcNAc glycosides carrying a linker for controlled coupling onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human beta1,4-galactosyltransferase-1 and beta1,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model galectin His(6)CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized surfaces by imitating the natural ECM microenvironment.
Herein is demonstrated that a biomimetic, in‐vivo‐like presentation of extracellular matrix (ECM) proteins such as fibronectin, laminin, and collagen IV mediated by a fully functional poly‐sugar (polyLacNAc)–lectin complex can be achieved on a biomaterials surface. Cells recognize and bind to these immobilized proteins and can, due to the reversible protein presentation, remodel initially presented ECM proteins.
Abstract:The enzymatic epimerization of uridine 5'-diphospho-a-d-glucose (UDP-Glc, 1) and uridine 5'-diphospho-N-acetyl-a-d-glucosamine (UDPGlcNAc, 2) and the subsequent oxidation of uridine 5'-diphospho-a-d-galactose (UDP-Gal, 3) and uridine 5'-diphospho-N-acetyl-a-d-galactosamine (UDP-GalNAc, 4) were combined with chemical biotinylation with biotin-e-amidocaproylhydrazide in a one-pot synthesis. Analysis by CE and NMR revealed a mixture (1.0:1.4) of the biotinylated nucleotide sugars uridine 5'-diphospho-6-biotin-e-amidocaproylhydrazino-a-d-galactose (UDP-6-biotinylGal, 7) and uridine 5'-diphospho-6-biotin-e-amidocaproylhydrazino-a-d-glucose (UDP-6-biotinyl-Glc, 9), respectively, in a reaction started with 1. One product, uridine 5'-diphospho-6-biotin-e-amidocaproylhydrazino-N-acetyl-a-d-galactosamine (UDP-6-biotinyl-GalNAc, 8) was formed when the reaction was initiated with 2. It could be demonstrated for the first time that a UDP-GlcA C H T U N G T R E N N U N G (NAc) 4'-epimerase (Gne from Campylobacter jejuni) and galactose oxidase from Dactylium dendroides can be used simultaneously in enzymatic catalysis. This is of particular interest since the coaction of an enzyme demanding reductive conditions and an oxygen-dependent oxidase is unexpected.
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