Molecular organization of the interferon gamma-binding domain in heparan sulphate.
Interferon (IFN)-gamma, in common with a number of cytokines or growth factors, strongly interacts with heparan sulphate (HS). It has been shown previously that one of the C-terminal basic clusters of amino acids (a regulatory element of IFN-gamma activity) is involved in this interaction. The structural organization of the HS domain that binds to human IFN-gamma has been investigated here. IFN-gamma-affinity chromatography 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-sulphated or N-acetylated. This led us to take a 'footprinting' approach in which HS was depolymerized in the presence of IFN-gamma and the cytokine-protected sequences were separated from the digested fragments. Using this strategy we consistently isolated an IFN-gamma-protected domain (IPD; approx. 10 kDa) which displayed the same affinity as full-length HS for the cytokine. Treatment of IPD with either heparinase or heparitinase strongly reduced its affinity, confirming that the high-affinity binding site encompassed a mixture of HS structural domains. Patterns of depolymerization with either enzymic or chemical agents were consistent with IPD being composed of an extended internal domain (approx. 7 kDa) which is predominantly N-acetylated and GlcA-rich, flanked by small N-sulphated oligosaccharides (mainly hexa- to octasaccharides). This is the first description of an HS protein-binding sequence with this type of molecular organization. Furthermore, using a cross-linking strategy, we demonstrated that one HS molecule bound to an IFN-gamma 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-gamma C-termini and bridge the two cytokine monomers through an internal N-acetyl-rich sequence.
Lortat-Jacob
H
H
Department of Cellular Pathology, Pasteur Institute, Centre National de la Recherche Scientifique URA 1459, Lyon, France.
Turnbull
J E
JE
Grimaud
J A
JA
D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases; D012867:Skin / Q000378:metabolism
D004187:Disaccharides; D006497:Heparan Sulfate; D007371:Interferon-gamma; D011133:Polysaccharide-Lyases; C021349:heparitinsulfate lyase