Interactions of N-acetyl-D-glucosamine-conjugated silk fibroin with lectins, cytoskeletal proteins and cardiomyocytes

Abstract

We have reported that cytoskeletal proteins such as desmin and vimentin are expressed on the surface of muscle, mesenchymal and cancer cells, and possess N-acetyl-β-D-glucosamine (β-GlcNAc) residue-binding properties. As cell-recognizable β-GlcNAc residue-bearing biopolymer, we prepared glycoconjugates (SF-GlcNAc) composed of silk fibroin (SF) and monosaccharide N-acetyl-D-glucosamine (GlcNAc) by chemical modification using cyanuric chloride. The covalent immobilization of GlcNAc into SF was assessed by 1H-NMR measurements. The 1H-NMR spectrum of SF-GlcNAc conjugates showed new peaks attributed to the methyl protons of the N-acetyl group in GlcNAc, and the integration of these peaks revealed that the GlcNAc content in the conjugates was 9 wt%. The existence of β-GlcNAc residues in SF-GlcNAc was examined by the criteria using lectins such as wheat germ agglutinin (WGA). Addition of WGA to SF-GlcNAc solution caused an increase in the turbidity of the solution due to lectin-mediated aggregation. Solid-phase lectin binding assay based on the biotin-avidin interaction showed that biotinylated succinylated WGA bound more strongly onto SF-GlcNAc conjugate-coated wells compared to SF-coated well. Following the establishment of the existence of β-GlcNAc residues in SF-GlcNAc, the interaction of SF-GlcNAc with desmin was examined by enzyme-linked immunosorbent assay using anti-desmin antibody. The stronger binding of desmin was observed for SF-GlcNAc conjugate-coated wells compared to SF-coated wells. The use of SF-GlcNAc conjugates as a substrate for culturing desmin-expressing human cardiac myocytes demonstrated an increase in the numbers of attached cells and proliferating cells on the conjugate-coated wells compared to SF-coated wells. These results suggest that the immobilization of monosaccharide GlcNAc is a useful method for the versatile functionalization of SF as an application in tissue engineering.