Chondroitin Sulfate Tetrasaccharides: Synthesis, Three-Dimensional Structure and Interaction with Midkine

The biological activity of midkine, a cytokine implicated in neuro- and tumourigenesis, is regulated by its binding to glycosaminoglycans (GAGs), such as heparin and chondroitin sulfate (CS). To better understand the molecular recognition of GAG sequences by this growth factor, the interactions between synthetic chondroitin sulfate-like tetrasaccharides and midkine were studied by using different techniques. Firstly, a synthetic approach for the preparation of CS-like oligosaccharides in the sequence GalNAc-GlcA was developed. A fluorescence polarisation competition assay was then employed to analyse the relative binding affinities of the synthetic compounds and revealed that midkine interacted with CS-like tetrasaccharides in the micromolar range. The 3D structure of these tetramers was studied in detail by a combination of NMR spectroscopy experiments and molecular dynamics simulations. Saturation transfer difference (STD) NMR spectroscopy experiments indicate that the CS tetrasaccharides bind to midkine in an extended conformation, with similar saturation effects along the entire sugar chain. These results are compatible with docking studies that suggest an interaction of the tetrasaccharide with midkine in a folded structure. Overall, this study provides valuable information on the interaction between midkine and well-defined, chemically synthesised CS oligosaccharides and these data can be useful for the design of more active compounds that modulate the biological function of this protein.