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.

The motional behaviour of heparin oligosaccharides in solution is best described as a top rotor having two perpendicular rotation axes. This prevents an accurate extraction of interprotonic distances by NOESY/ROESY based methods. In this paper, we describe the solution structure of the hexasaccharide 1 calculated from high exactitude distance data obtained from off-resonance ROESY combined with a long MD simulation of 500 ns. In previous studies, we have found that two synthetic hexasaccharides having the sulphate groups directed towards one side of its central plane have an opposite biological activity, while 1 is unable to activate the FGF-1 signalling pathway, the other (2) is even more active than the regular region derived hexasaccharide (3) that mimics the natural active compound, heparin. From the structural analysis it was concluded that 1 has similar three-dimensional characteristics to 2 or 3 and therefore the differences in the activity should be due to the arrangement of the sulphate groups within the hexasaccharidic sequence.