Investigation of the Influence Glycosaminoglycan Sulfation on the Interaction with Interleukin-8 by Fluorescence and Solution NMR Spectroscopy

Abstract

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix (ECM), and proteins such as growth factors and chemokines play a critical role in cellular regulation processes. Previous work on the chemokine Interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. Nevertheless, low or non sulfated GAGs such as hyaluronan (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), also contained in the extracellular matrix, have so far not been studied with regard to their distinct binding properties towards IL-8.In this work, we combine fluorescence as well as solution NMR experiments to study the recognition properties of GAG hexasaccharides, including HA, CS, DS and heparin, to IL-8. Using 1H-15N HSQC spectroscopy of IL-8 in the presence of varying GAG concentrations, the interacting amino acid could be identified. Furthermore to observe lysine side chains, a reductive methylation with [13C]-formaldehyde, adding two methyl groups to the e-NH3+ of lysines without altering the positive charge, was performed. In order to resolve the signals from the lysine side chains, 1H-13C HSQC spectra were recorded and the influence of the GAGs was tested again.The results show that an increase in GAG sulfation enhances the strength of the binding to the protein. In addition, in cases of equal degree of sulfation the position of the sulfate group plays a crucial role sugessting that interactions between proteins and GAGs are not purely electrostatically driven and steric forces as well as contributions from hydrogen bonds have to be considered. These observations were also confirmed by molecular docking and dynamic simulations providing a structural picture of the interaction of IL-8 with various GAGs.