Hepatocyte growth factor/scatter factor binds to small heparin-derived oligosaccharides and stimulates the proliferation of human HaCaT keratinocytes.
Hepatocyte growth factor/scatter factor (HGF/SF) acts via a dual receptor system consisting of the MET tyrosine kinase receptor and heparan sulfate or dermatan sulfate proteoglycans. In optical biosensor binding assays, competition by oligosaccharides for binding of HGF/SF to immobilized heparin showed that disaccharides failed to compete, whereas tetrasaccharides inhibited HGF/SF binding (IC(50) 8 microg/ml). The inhibitory potency of the oligosaccharides increased as their length increased by successive disaccharide units, to reach a maximum (IC(50) 1 microg/ml) at degree of polymerization (dp) 10. In binding assays, HGF/SF was found to bind directly to oligosaccharides as small as dp 4, and the binding parameters were similar for oligosaccharides of dp 4-14 (k(a) 2.2-45.3 x 10(6) m(-1) s(-1), k(d) 0.033-0.039 s(-1), and K(d) 9-16 nm). In human keratinocytes, HGF/SF stimulated DNA synthesis, and this was dependent on a sustained phosphorylation of p42/44(MAPK). In chlorate-treated and hence sulfated glycosaminoglycan-deficient HaCaT cells, the stimulation of DNA synthesis by HGF/SF was almost abolished. Heparin-derived oligosaccharides from dp 2 to dp 24 were added together with HGF/SF to chlorate-treated cells to determine the minimum size of oligosaccharides able to restore HGF/SF activity. At restricted concentrations of oligosaccharides (4 ng/ml), HGF/SF required decasaccharides, whereas at higher concentrations (100 ng/ml) even tetrasaccharides were able to partly restore DNA synthesis. The results suggest that HGF/SF binds to a tetrasaccharide and that although this is sufficient to enable the stimulation of DNA synthesis, longer oligosaccharides are more efficient, perhaps by virtue of their ability to bind more easily other molecules.
Delehedde
Maryse
M
School of Biological Sciences, Life Science Building, University of Liverpool, Crown Street, Liverpool L69 7ZB, United Kingdom.
Lyon
Malcolm
M
Vidyasagar
Rishma
R
McDonnell
Timothy J
TJ
Fernig
David G
DG
D001667:Binding, Competitive; D015374:Biosensing Techniques; D019921:Biotinylation; D002455:Cell Division; D002704:Chlorates / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006025:Glycosaminoglycans / Q000737:chemistry; D006493:Heparin / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D015603:Keratinocytes / Q000378:metabolism*; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors
D002704:Chlorates; D006025:Glycosaminoglycans; D009844:Oligosaccharides; D017228:Hepatocyte Growth Factor; D006493:Heparin; D004247:DNA; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases