title: Hairpin loop and second kringle domain are essential sites for heparin binding and biological activity of hepatocyte growth factor.
abstract: Hepatocyte growth factor (HGF) has a strong affinity for heparin. About one fourth of HGF secreted from MRC-5 human embryonic lung fibroblast cells was found to be associated with heparin and heparan sulfate proteoglycan on the cell surface and extracellular matrix. To identify heparin-binding sites within the HGF molecule, we constructed variously deleted mutant HGFs and examined their binding ability to an immobilized heparin column. Native HGF and mutant HGFs, including d-K1 (deletion of the first kringle domain), d-K3 (deletion of the third kringle domain), d-K4 (deletion of the fourth kringle domain), d-beta (deletion of beta-chain), and HK1K2 (consisting of the N-terminal hairpin loop and the first two kringle domains), tightly bound to a heparin column, but d-H (deletion of the N-terminal hairpin loop) and d-K2 (deletion of the second kringle domain) markedly decreased binding ability to the column. These observations suggest that the N-terminal hairpin loop and the second kringle domain are essential for the heparin-binding of HGF. The finding that HK1K2 competed the binding of 125I-HGF to immobilized heparin provided additional evidence that the N-terminal half of HGF alpha-chain is the principal heparin-binding site. The hairpin loop in HGF possesses a cluster of basic amino acid residues and a highly positive net charge, when compared with hairpin loop structures in the other proteins, plasminogen and HGF-like protein. The second kringle domain in HGF has the basic amino acid cluster in the central region. Thus, it is likely that the basic clusters in these domains cooperatively contribute to the binding of HGF to the anionic heparin or heparan sulfate molecule.
authors.affiliation: Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Mizuno
authors.forename: H
authors.initials: H
authors.lastname: Inoue
authors.forename: M
authors.initials: M
authors.lastname: Hagiya
authors.forename: S
authors.initials: S
authors.lastname: Shimizu
authors.forename: T
authors.initials: T
authors.lastname: Nose
authors.forename: Y
authors.initials: Y
authors.lastname: Shimohigashi
authors.forename: T
authors.initials: T
authors.lastname: Nakamura
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000737:chemistry* / Q000378:metabolism; D018082:Kringles; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate; D017228:Hepatocyte Growth Factor; D011509:Proteoglycans; D019859:Proto-Oncogene Proteins c-met; D020794:Receptor Protein-Tyrosine Kinases