The immune response against microbial infections bears the intrinsic risk of an immune-mediated inflammatory damage to the host tissue. The cytokine interleukin (IL)-10 is a key regulator of the innate and adaptive immune system, which prevents an overwhelming immune reaction and tissue damage1. IL-10 inhibits the synthesis of pro-inflammatory cytokines (IFNγ, TNFα) and of cell surface molecules (MHC class II proteins). Thereby, cellular immune responses mediated by macrophages or T cells are inactivated. IL-10 has mostly paracrine functions and acts over short distances within the tissue2. In this context, glycosaminoglycans (GAGs) of the extracellular matrix have been suggested as important binding partners of IL-10 that restrict the protein to the vicinity of the secreting or targeting cell3. GAGs are a class of highly sulfated carbohydrate molecules that are known to bind and regulate a number of distinct proteins, including chemokines, cytokines, growth factors and adhesion molecules4.
Here, we have analyzed the molecular interactions between IL-10 and different GAG oligosaccharides by NMR spectroscopy. Chemical shift perturbations from [1H-15N]-HSQC spectra were used to identify the GAG binding site within IL-10. Binding occurs mainly at the DE-loop, which forms a crevice between both subunits of the IL-10 dimer. Additional contacts are made with residues of the N- and C-terminus. Furthermore, saturation transfer difference (STD) NMR spectroscopy revealed the important carbohydrate binding epitopes and could quantify binding affinity of different oligosaccharides. In particular, N-sulfation of the GAG hexosamine unit is critical for interaction with IL-10. Upon binding the carbohydrate adopts a slightly bended conformation as indicated by transfer NOESY NMR experiments. For heparin, additional NOEs are observed between proton H2 of glucosamine and H1 of the respective preceding iduronic acid unit. At the end, our data help to understand how biological functions of IL-10 are related to its interaction with extracellular matrix GAGs.