Carboxyl‐terminal structure of basic fibroblast growth factor significantly contributes to its affinity for heparin

Abstract

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 Asp4' and SerlOO. Moreover, it suggests that the Phe139Leu'40Pro141, present in all members of the FGF family, contributes greatly to the stable structure of the intact molecule. . Extraordinarily high affinity for heparin is one of the important characteristics of this family. In fact, this characteristic has been applied to the isolation and characterization of these proteins, especially basic FGF (bFGF) and acidic FGF (aFGF), from various tissues [15] and tumors [16]. The identification of specific fundamental domains of bFGF for receptor and heparin binding has been reported using synthetic polypeptides; these have suggested the existence of two domains for heparin binding and one for receptor binding [I 5 , 171. We isolated the cDNA for human bFGF and studied the expression of this cDNA in mammalian cells [18, 191 and in bacterial cells (201. We also purified and characterized the recombinant bFGF produced in Escherichia coli [20] and studied the contribution of the four cysteine residues to the protein conformation of bFGF through a site-directed mutagenesis procedure [21]. For further characterization, we constructed several bFGF molecules whose amino-or carboxyl-terminus was deleted. Here we report the structural characterization for heparin binding using these terminally truncated molecules.Correspondence to M . Seno, Biotechnology Research Laboratories, Research and Development Div., Takeda Chemical Industries Ltd, 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532, JapanAbbreviations. F G F , fibroblast growth factor; bFGF, basic fibroblast growth factor; aFGF, acidic fibroblast growth factor.Enzymes. DNA polymerase I (EC 2.7.7.7); exonucleasc I11 (EC 3.1.11.2); lysozyme (EC 3.2.1.17); peroxidase (EC 1.11.1.7). MA...

Isolation of peptides that inhibit binding of basic fibroblast growth factor to its receptor from a random phage-epitope library.

“…Moreover, high-affinity receptor binding of FGF requires heparin or cell-surface heparan sulfates (11,12). Several attempts have been made to identify functional domains in bFGF that participate in receptor binding and could serve as bFGF antagonists (27,28). In an extensive survey of overlapping synthetic peptides spanning the entire primary sequence of bFGF, Baird and colleagues (27) identified two major regions suspected of involvement in ligand-receptor interaction.…”

Glu-96 of Basic Fibroblast Growth Factor Is Essential for High Affinity Receptor Binding

“…Later, studies of the three-dimensional structure of bFGF revealed that part of this putative receptor binding peptide forms a loop consisting of residues 109 -114 on the surface of the molecule (Eriksson et al, 1993). In addition, Seno et al (1990) studied the action of truncated N-and C-terminal forms of bFGF on mitogenesis and heparin binding. Their results indicate that an essential part of bFGF for receptor binding is present within the sequence Asp-41 to Ser-100.…”

“…Cultures were grown to an A 600 of 0.8 in LB medium containing 40 g/ml ampicillin at 37°C, and bFGF expression was induced by adding 0.4 mM isopropyl-␤-D-thiogalactopyranoside to further culture at 37°C in a thermoshaker for 4 h. The bFGF containing cell culture was then pelleted by centrifugation and lysed according to standard procedures (Seno et al, 1990). The cytoplasmic fraction containing the mutein was loaded onto a heparin-Sepharose column equilibrated with buffer containing 0.6 M NaCl, 25 mM Tris, pH 7.5.…”

Nonenzymatic glycosylation in vitro and in bovine endothelial cells alters basic fibroblast growth factor activity. A model for intracellular glycosylation in diabetes.

“…If (Fig. 6) Asn'"' to Prol4l, although truncation of even seven amino acid residues from the carboxyl-terminus (including Lys'45) significantly reduces bFGF's affinity for heparin (63). In contrast, the mitogenic receptor binding activity of bFGF appears to be associated with two other regions of the molecule, amino acid residues and 102-129 (64).…”