Engineered mutants of HGF/SF with reduced binding to heparan sulphate proteoglycans, decreased clearance and enhanced activity in vivo.
BACKGROUND
Although a number of growth factors bind cell-surface heparan sulphate proteoglycans (HSPGs), the role of this interaction is unclear except for fibroblast growth factor which requires HSPG binding for signalling. Hepatocyte growth factor/scatter factor (HGF/SF) plays important roles in mammalian development and tissue regeneration and acts on target cells through a specific receptor tyrosine kinase encoded by the c-met proto-oncogene. This factor also binds HSPGs with high affinity, but conflicting data have been reported on the role of HSPG binding in HGF/SF signalling.


RESULTS
To map the binding sites for HSPG and the Met receptor in HGF/SF, we have engineered a number of HGF/SF mutants in which several clusters of solvent-accessible residues in the hairpin structure of the amino-terminal domain or in kringle 2 have been replaced. Two of the mutants (HP1 and HP2) showed greatly decreased (more than 50-fold) affinity for heparin and HSPGs but retained full mitogenic and motogenic activities on target cells in culture. Furthermore, when compared with wild-type HGF/SF, the HP1 mutant exhibited a delayed clearance from the blood, higher tissue levels and a higher induction of DNA synthesis in normal, adult murine liver.


CONCLUSIONS
These results establish the following: the binding sites in HGF/SF for Met and for HSPGs can be dissociated by protein engineering; high-affinity binding of HGF/SF to HSPGs is not essential for signalling; one role of HSPG binding in the HGF/SF system appears to be sequestration and degradation of the growth factor; and HGF/SF mutants with decreased affinity for HSPGs exhibit enhanced activity in vivo.
Hartmann
G
G
Imperial Cancer Research Fund Laboratory, MRC Centre, Hills Road, Cambridge, CB2 2QH, UK.
Prospero
T
T
Brinkmann
V
V
Ozcelik
C
C
Winter
G
G
Hepple
J
J
Batley
S
S
Bladt
F
F
Sachs
M
M
Birchmeier
C
C
Birchmeier
W
W
Gherardi
E
E
Ozcelik
O
O
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004261:DNA Replication / Q000187:drug effects; D004285:Dogs; D005260:Female; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D006801:Humans; D018082:Kringles / Q000235:genetics; D008099:Liver / Q000378:metabolism; D008657:Metabolic Clearance Rate; D008907:Mink; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D000090063:Proto-Oncogene Mas; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D014018:Tissue Distribution
D019812:Heparan Sulfate Proteoglycans; C000715470:MAS1 protein, human; D010446:Peptide Fragments; D000090063:Proto-Oncogene Mas; D011993:Recombinant Fusion Proteins; D017228:Hepatocyte Growth Factor; D006493:Heparin; D019859:Proto-Oncogene Proteins c-met