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.
Full-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.
Cancer Research UK, University of Manchester, Christie Hospital National Health Service Trust, Wilmslow Road, M20 4BX. MLyon@picr.man.ac.uk
Malcolm
M
Lyon
Jon A
JA
Deakin
Daniel
D
Lietha
Ermanno
E
Gherardi
John T
JT
Gallagher
D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004789:Enzyme Activation; D014644:Genetic Variation; D006025:Glycosaminoglycans / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009101:Multiple Myeloma; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion
D006025:Glycosaminoglycans; D009844:Oligosaccharides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases