ENGINEERED MUTANTS OF HGF SF WITH REDUCED BINDING TO HEPARAN-SULFATE PROTEOGLYCANS, DECREASED CLEARANCE AND ENHANCED ACTIVITY IN-VIVO/

Background: Although a number of growth factors bind cell-surface hepa ran sulphate proteoglycans (HSPGs), the role of this interaction is un clear 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 encod ed by the c-met protooncogene. 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 HS PG 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 ha ve been replaced. Two of the mutants (HP1 and HP2) showed greatly decr eased (more than 50-fold) affinity for heparin and HSPGs but retained full mitogenic and motogenic activities on target cells in culture. Fu rthermore, when compared with wild-type HGF/SF, the HPI mutant exhibit ed a delayed clearance from the blood, higher tissue levels and a high er induction of DNA synthesis in normal, adult murine liver. Conclusio ns: 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 sequestrat ion and degradation of the growth factor; and HGF/SF mutants with decr eased affinity for HSPGs exhibit enhanced activity in vivo. (C) Curren t Biology Ltd.