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dc.contributor.authorLyon, Malcolm
dc.contributor.authorDeakin, Jon A
dc.contributor.authorLietha, Daniel
dc.contributor.authorGherardi, Ermanno
dc.contributor.authorGallagher, John T
dc.date.accessioned2009-08-24T16:27:48Z
dc.date.available2009-08-24T16:27:48Z
dc.date.issued2004-10-15
dc.identifier.citationThe interactions of hepatocyte growth factor/scatter factor and its NK1 and NK2 variants with glycosaminoglycans using a modified gel mobility shift assay. Elucidation of the minimal size of binding and activatory oligosaccharides. 2004, 279 (42):43560-7 J. Biol. Chem.en
dc.identifier.issn0021-9258
dc.identifier.pmid15292253
dc.identifier.doi10.1074/jbc.M408510200
dc.identifier.urihttp://hdl.handle.net/10541/78398
dc.description.abstractFull-length hepatocyte growth factor/scatter factor interacts with both heparan and dermatan sulfates and is critically dependent upon them as cofactors for activation of the tyrosine kinase receptor Met. Two C-terminally truncated variants (NK1 and NK2) of this growth factor also occur naturally. Their glycosaminoglycan binding properties are not clear. We have undertaken a comparative study of the heparan/dermatan sulfate binding characteristics of all three proteins. This has entailed the development of a modified gel mobility shift assay, utilizing fluorescence end-tagged oligosaccharides, that is also widely applicable to the analysis of many glycosaminoglycan-protein interactions. Using this we have shown that all three hepatocyte growth factor/scatter factor variants share identical heparan/dermatan sulfate binding properties and that both glycosaminoglycans occupy the same binding site. The minimal size of the oligosaccharide that binds with high affinity in all cases is a tetrasaccharide from heparan sulfate but a hexasaccharide from dermatan sulfate. These findings demonstrate that functional glycosaminoglycan binding is restricted to a binding site situated solely within the small N-terminal domain. The same minimal size fractions are also able to promote hepatocyte growth factor/scatter factor-mediated activation of Met and consequent downstream signaling in the glycosaminoglycan-deficient Chinese hamster ovary pgsA-745 cells. A covalent complex of heparan sulfate tetrasaccharide with monovalent growth factor is also active. The binding and activity of tetrasaccharides put constraints upon the possible interactions and molecular geometry within the ternary signaling complex.
dc.language.isoenen
dc.subjectCell Line Tumouren
dc.subject.meshCell Line, Tumor
dc.subject.meshCloning, Molecular
dc.subject.meshEnzyme Activation
dc.subject.meshGenetic Variation
dc.subject.meshGlycosaminoglycans
dc.subject.meshHepatocyte Growth Factor
dc.subject.meshHumans
dc.subject.meshKinetics
dc.subject.meshMitogen-Activated Protein Kinases
dc.subject.meshMultiple Myeloma
dc.subject.meshOligosaccharides
dc.subject.meshPichia
dc.subject.meshProtein-Serine-Threonine Kinases
dc.subject.meshProto-Oncogene Proteins
dc.subject.meshProto-Oncogene Proteins c-akt
dc.subject.meshProto-Oncogene Proteins c-met
dc.subject.meshRecombinant Proteins
dc.subject.meshSequence Deletion
dc.titleThe interactions of hepatocyte growth factor/scatter factor and its NK1 and NK2 variants with glycosaminoglycans using a modified gel mobility shift assay. Elucidation of the minimal size of binding and activatory oligosaccharides.en
dc.typeArticleen
dc.contributor.departmentCancer Research UK, University of Manchester, Christie Hospital National Health Service Trust, Wilmslow Road, M20 4BX. MLyon@picr.man.ac.uken
dc.identifier.journalThe Journal of Biological Chemistryen
html.description.abstractFull-length hepatocyte growth factor/scatter factor interacts with both heparan and dermatan sulfates and is critically dependent upon them as cofactors for activation of the tyrosine kinase receptor Met. Two C-terminally truncated variants (NK1 and NK2) of this growth factor also occur naturally. Their glycosaminoglycan binding properties are not clear. We have undertaken a comparative study of the heparan/dermatan sulfate binding characteristics of all three proteins. This has entailed the development of a modified gel mobility shift assay, utilizing fluorescence end-tagged oligosaccharides, that is also widely applicable to the analysis of many glycosaminoglycan-protein interactions. Using this we have shown that all three hepatocyte growth factor/scatter factor variants share identical heparan/dermatan sulfate binding properties and that both glycosaminoglycans occupy the same binding site. The minimal size of the oligosaccharide that binds with high affinity in all cases is a tetrasaccharide from heparan sulfate but a hexasaccharide from dermatan sulfate. These findings demonstrate that functional glycosaminoglycan binding is restricted to a binding site situated solely within the small N-terminal domain. The same minimal size fractions are also able to promote hepatocyte growth factor/scatter factor-mediated activation of Met and consequent downstream signaling in the glycosaminoglycan-deficient Chinese hamster ovary pgsA-745 cells. A covalent complex of heparan sulfate tetrasaccharide with monovalent growth factor is also active. The binding and activity of tetrasaccharides put constraints upon the possible interactions and molecular geometry within the ternary signaling complex.


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