Site-directed mutagenesis and molecular modeling identify a crucial amino acid in specifying the heparin affinity of FGF-1.
Heparin potentiates the mitogenic activity of FGF-1 by increasing the affinity for its receptor and by extending its biological half-life. During the course of labeling human FGF-1 with Na(125)I and chloramine T, it was observed that the protein lost its ability to bind to heparin. In contrast, bovine FGF-1 retained its heparin affinity even after iodination. To localize the region responsible for the lost heparin affinity, chimeric FGF-1 proteins were constructed from human and bovine FGF-1 expression constructs and tested for their heparin affinity after iodination. The results showed that the C-terminal region of human FGF-1 was responsible for the loss of heparin affinity. This region harbors a single tyrosine residue in human FGF-1 in contrast to a phenylalanine at this position in bovine FGF-1. Mutating this tyrosine residue in the human FGF-1 sequence to phenylalanine did not restore the heparin affinity of the iodinated protein. Likewise, changing the phenylalanine to tyrosine in the bovine FGF-1 did not reduce the ability of the iodinated protein to bind to heparin. In contrast, a mutant human FGF-1 that has cysteine-131 replaced with serine (C131S) was able to bind to heparin even after iodination while bovine FGF-1 (S131C) lost its binding affinity to heparin upon iodination. In addition, the human FGF-1 C131S mutant showed a decrease in homodimer formation when exposed to CuCl(2). Molecular modeling showed that the heparin-binding domain of FGF-1 includes cysteine-131 and that cysteine-131, upon oxidation to cysteic acid during the iodination procedures, would interact with lysine-126 and lysine-132. This interaction alters the conformation of the basic residues such that they no longer bind to heparin.
Patrie
K M
KM
Department of Internal Medicine, Davis Medical Research Center, The Ohio State University, Columbus 43210, USA.
Botelho
M J
MJ
Franklin
K
K
Chiu
I M
IM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D003544:Cysteic Acid / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000502:physiology; D016220:Fibroblast Growth Factor 1; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D000596:Amino Acids; D007457:Iodine Radioisotopes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D006493:Heparin; D003544:Cysteic Acid; D003545:Cysteine