Abstract:The biological activity of midkine, a cytokine implicated in neurogenesis and tumorigenesis, 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, we have studied here the interactions between synthetic chondroitin sulfate-like tetrasaccharides and midkine using different techniques. First, a synthetic approach for the preparation of CS-like oligosaccharides in the sequence GalNAc-GlcA was developed. A fluorescence polarization competition assay was then employed to analyse the relative binding affinities of the synthetic compounds and revealed that midkine interacts with CS-like tetrasaccharides in the micromolar range. The 3D structure of these tetramers was studied in detail by a combination of NMR experiments and molecular dynamics simulations. Saturation transfer difference (STD) NMR 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 suggesting an interaction of the tetrasaccharide with midkine in a folded structure. Overall, our study gives valuable information on the interaction between midkine and well-defined, chemically synthesized CS oligosaccharides and these data can be useful for the design of more active compounds that modulate the biological function of this protein.

Here, we present a novel approach for the chemical synthesis of chondroitin and dermatan sulfate oligosaccharides. A key point of this strategy is the preparation and use of an N-trifluoroacetyl galactosamine building block containing a 4,6-O-di-tert-butylsilylene group. Glycosylation reactions proceeded in good yields (74-91%) with our protecting group distribution. Using this approach, we have synthesized, for the first time, a chondroitin/dermatan sulfate-like tetrasaccharide that contains both types of uronic acids, D-glucuronic and L-iduronic acid. Moreover, we have employed a fluorescence polarization competition assay to evaluate the interactions between the synthesized oligosaccharides and FGF-2 (basic fibroblast growth factor). Our results show that this method, using standard instrumentation and minimal sample consumption, is a powerful tool for the rapid analysis of the glycosaminoglycan affinity for proteins in solution.