Carboxyl-terminal structure of basic fibroblast growth factor significantly contributes to its affinity for heparin.
The carboxyl-terminal sequence of basic fibroblast growth factor (bFGF) is rich in basic amino acid residues, a common characteristic amongst fibroblast growth factors, and is considered to contribute greatly to the binding to negatively charged extracellular matrixes such as heparin. To study the relationship between the affinity for heparin and the carboxyl-terminal structure of bFGF, amino- or carboxyl-terminal truncated molecules were produced in Escherichia coli using recombinant DNA techniques. These terminally truncated bFGFs were applied to a heparin-affinity HPLC column. Truncation of more than six amino acid residues from the carboxyl-terminal made the bFGF produced in E. coli markedly difficult to solubilize and weakened its affinity for heparin, though bFGF having up to 46 amino acids removed showed significant stimulation of the DNA synthesis of BALB/c3T3 cells. This stimulation of the DNA synthesis was also recognized by the bFGF having 40 amino acids removed from its amino-terminal, while the affinity of this peptide for heparin has been shown to be equal to that of the mature bFGF (146 amino acids). These results show that the affinity of bFGF for heparin depends significantly on its carboxyl-terminal structure and that the essential part for receptor binding is present between Asp41 and Ser100. Moreover, it suggests that the Phe139Leu140Pro141, present in all members of the FGF family, contributes greatly to the stable structure of the intact molecule.
Seno
M
M
Biotechnology Research Laboratories, Takeda Chemical Industries Ltd, Osaka, Japan.
Sasada
R
R
Kurokawa
T
T
Igarashi
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D005346:Fibroblast Growth Factors; D006493:Heparin; D004247:DNA