Molecular organization of the interferon gamma-binding domain in heparan sulphate.

2.50
Hdl Handle:
http://hdl.handle.net/10541/97493
Title:
Molecular organization of the interferon gamma-binding domain in heparan sulphate.
Authors:
Lortat-Jacob, H; Turnbull, Jeremy E; Grimaud, J A
Abstract:
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.
Affiliation:
Department of Cellular Pathology, Pasteur Institute, Centre National de la Recherche Scientifique URA 1459, Lyon, France.
Citation:
Molecular organization of the interferon gamma-binding domain in heparan sulphate. 1995, 310 ( Pt 2):497-505 Biochem. J.
Journal:
Biochemical Journal
Issue Date:
1-Sep-1995
URI:
http://hdl.handle.net/10541/97493
PubMed ID:
7654188
Type:
Article
Language:
en
ISSN:
0264-6021
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorLortat-Jacob, Hen
dc.contributor.authorTurnbull, Jeremy Een
dc.contributor.authorGrimaud, J Aen
dc.date.accessioned2010-04-27T14:58:36Z-
dc.date.available2010-04-27T14:58:36Z-
dc.date.issued1995-09-01-
dc.identifier.citationMolecular organization of the interferon gamma-binding domain in heparan sulphate. 1995, 310 ( Pt 2):497-505 Biochem. J.en
dc.identifier.issn0264-6021-
dc.identifier.pmid7654188-
dc.identifier.urihttp://hdl.handle.net/10541/97493-
dc.description.abstractInterferon (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.en
dc.language.isoenen
dc.subject.meshAnimals-
dc.subject.meshBinding Sites-
dc.subject.meshCarbohydrate Sequence-
dc.subject.meshCattle-
dc.subject.meshCells, Cultured-
dc.subject.meshChromatography, Affinity-
dc.subject.meshChromatography, Gel-
dc.subject.meshChromatography, High Pressure Liquid-
dc.subject.meshDisaccharides-
dc.subject.meshFibroblasts-
dc.subject.meshHeparitin Sulfate-
dc.subject.meshHumans-
dc.subject.meshInterferon-gamma-
dc.subject.meshMolecular Sequence Data-
dc.subject.meshPolysaccharide-Lyases-
dc.subject.meshSkin-
dc.titleMolecular organization of the interferon gamma-binding domain in heparan sulphate.en
dc.typeArticleen
dc.contributor.departmentDepartment of Cellular Pathology, Pasteur Institute, Centre National de la Recherche Scientifique URA 1459, Lyon, France.en
dc.identifier.journalBiochemical Journalen
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