MOLECULAR-ORGANIZATION OF THE INTERFERON GAMMA-BINDING DOMAIN IN HEPARAN-SULFATE

Interferon (IFN)-gamma, in common with a number of cytokines or growth factors, strongly interacts with heparan sulphate (HS). It has been s hown previously that one of the C-terminal basic clusters of amino aci ds (a regulatory element of IFN-gamma activity) is involved in this in teraction. The structural organization of the HS domain that binds to human IFN-gamma has been investigated here. IFN-gamma-affinity chromat ography of HS oligosaccharides released by either enzymic or chemical cleavage showed that the binding site is not found in a domain that is resistant to either heparinase or heparitinase or exclusively N-sulph ated. or N-acetylated. This led us to take a 'footprinting' approach i n which HS was depolymerized in the presence of IFN-gamma and the cyto kine-protected sequences were separated from the digested fragments. U sing this strategy we consistently isolated an IFN-gamma-protected dom ain (IPD; approx. 10 kDa) which displayed the same affinity as full-le ngth HS for the cytokine. Treatment of IPD with either heparinase or h eparitinase strongly reduced its affinity, confirming that the high-af finity binding site encompassed a mixture of HS structural domains. Pa tterns of depolymerization with either enzymic or chemical agents were consistent with IPD being composed of an extended internal domain (ap prox. 7kDa) which is predominantly N-acetylated and GlcA-rich, Banked by small N-sulphated oligosaccharides (mainly hexa- to octasaccharides ). This is the first description of an HS protein-binding sequence wit h this type of molecular organization. Furthermore, using a cross-link ing strategy, we demonstrated that one HS molecule bound to an IFN-gam ma dimer. Together these results lead us to propose a novel model for the interaction of HS with a protein, in which two sulphated terminal sequences of the binding domain interact directly with the two IFN-gam ma C-termini and bridge the two cytokine monomers through an internal N-acetyl-rich sequence.