The 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.
AffiliationCancer Research UK, University of Manchester, Christie Hospital National Health Service Trust, Wilmslow Road, M20 4BX. MLyon@picr.man.ac.uk
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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.
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.
JournalThe Journal of Biological Chemistry
- The mode of action of heparan and dermatan sulfates in the regulation of hepatocyte growth factor/scatter factor.
- Authors: Lyon M, Deakin JA, Gallagher JT
- Issue date: 2002 Jan 11
- The binding properties of minimal oligosaccharides reveal a common heparan sulfate/dermatan sulfate-binding site in hepatocyte growth factor/scatter factor that can accommodate a wide variety of sulfation patterns.
- Authors: Deakin JA, Blaum BS, Gallagher JT, Uhrín D, Lyon M
- Issue date: 2009 Mar 6
- Hepatocyte growth factor/scatter factor binds to small heparin-derived oligosaccharides and stimulates the proliferation of human HaCaT keratinocytes.
- Authors: Delehedde M, Lyon M, Vidyasagar R, McDonnell TJ, Fernig DG
- Issue date: 2002 Apr 5
- Differential regulation of hepatocyte growth factor/scatter factor by cell surface proteoglycans and free glycosaminoglycan chains.
- Authors: Deakin JA, Lyon M
- Issue date: 1999 Jun
- Dissociation of heparan sulfate and receptor binding domains of hepatocyte growth factor reveals that heparan sulfate-c-met interaction facilitates signaling.
- Authors: Rubin JS, Day RM, Breckenridge D, Atabey N, Taylor WG, Stahl SJ, Wingfield PT, Kaufman JD, Schwall R, Bottaro DP
- Issue date: 2001 Aug 31