NMR characterization of the binding properties and conformation of glycosaminoglycans interacting with interleukin-10

Glycobiology. 2014 Nov;24(11):1036-49. doi: 10.1093/glycob/cwu069. Epub 2014 Jul 6.

Abstract

The cytokine interleukin-10 (IL-10) is an important regulator of the host immune system with both pro- and anti-inflammatory functions. Glycosaminoglycans (GAGs) play a decisive role in the biology of many growth factors, e.g., for receptor binding or protection from proteolytic degradation. GAGs of the extracellular matrix inhibit IL-10 signaling, however, the molecular mechanism is so far unknown. Here, we studied the interaction between GAGs and IL-10 using a combination of nuclear magnetic resonance (NMR) spectroscopy and computer simulations. The binding region of a set of heparin and chondroitin sulfate GAG disaccharides with varying sulfation pattern were determined by saturation transfer difference (STD) NMR spectroscopy. From the initial growth rate of the STD amplification factor binding affinities were determined and KD values in the low millimolar to micromolar range were obtained. We observed the highest binding affinity to IL-10 with fully sulfated heparin; however, a hyaluronan hexasaccharide did not exhibit binding, which suggests that GAG sulfation is necessary for interaction with IL-10. For octasaccharides or longer GAGs, a cooperative binding behavior was observed, which could indicate simultaneous interaction with both dimer subunits of IL-10. Finally, structural information about the bound GAG was exemplarily obtained for a heparin tetrasaccharide fragment (ΔUA,2S-GlcNS,6S-IdoA,2S-GlcNS,6S) using transferred NOESY experiments, proton-proton scalar couplings and molecular dynamics simulations. The overall backbone conformation is only slightly changed in the presence of IL-10 and the conformational equilibrium between (1)C4 chair and (2)So skew-boat structure of the internal iduronic acid residue is preserved.

Keywords: NOE; STD spectroscopy; glycosaminoglycans; interleukin-10; molecular dynamics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Glycosaminoglycans / metabolism*
  • Interleukin-10 / metabolism*
  • Magnetic Resonance Spectroscopy / methods*
  • Mice
  • Molecular Dynamics Simulation

Substances

  • Glycosaminoglycans
  • Interleukin-10