Andrew P. Seddon

Isolation from Bovine Brain and Structural Characterization of HBNF, a Heparin-Binding Neurotrophic Factor

A heparin-binding protein with neurotrophic activity for perinatal rat neurons, termed HBNF, was purified to homogeneity from bovine brain utilizing pH 4.5 extraction, ammonium sulfate precipitation, cation exchange and heparin-Sepharose affinity chromatographies, and reverse phase HPLC. In the presence of protease inhibitors during extraction, a protein with an apparent molecular weight of 18 kDa was obtained in a yield of approximately 0.5 mg/kg brain tissue. The amino acid sequence of the first 114 residues of HBNF was determined and found to highly homologous to the cDNA-derived amino acid sequence of human HBNF, a 136-residue protein. Bovine and human HBNFs have identical molecular weights as judged by SDS gel electrophoresis and very similar amino acid compositions. This and overall sequence conservation suggest that bovine HBNF is also a 136 amino acid protein with a calculated molecular weight of approximately 15.5 kDa. The apparent discrepancy between calculated and observed molecular weights of bovine HBNF (and of human HBNF of which the complete sequence is known) is most likely a result of the highly basic nature of HBNF. If protease inhibitors were omitted during tissue extraction, two additional proteins with lower apparent molecular weights and identical N-terminal sequences were isolated, with the smallest forms being the major product. Amino acid analysis showed that the smaller forms correspond to C-terminally truncated HBNFs with calculated molecular weights of 13.6 and 12.4 kDa, lacking approximately 14 and 22 residues. Comparison of the HBNF protein sequence with sequences stored in the Protein Identification Resource/Genbank databases reveals high homology to the translation product of the MK-1 gene, which is retinoic acid-inducible in embryonic carcinoma cells and developmentally expressed during gestation in mice.

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

SummaryThe assignments of the 1H, 15N, 13CO and 13C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of Cα, Cβ and Hα to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of Cα, Cβ and Hα to the backbone amide 1H and 15N in the CBCANH and HNHA experiments. In addition, Cα and Cβ chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and Cα correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and Cα correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic 1H and 13C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, Hα and Hβ protons as well as 3JHnHα coupling constants, amide exchange and 13Cα and 13Cβ secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel β-sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as β-strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the β-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.

Structure/Activity Relationships in Basic FGF

Although the FGFs have been subject to extensive biological studies, only limited progress has been made so far in determining the critical elements of structure-activity relationships in the FGFs. Among the recognized structural elements with potential to affect the biological activity of FGFs are the cysteine residues, and the heparin- and receptor-binding domains. These features have been studied using a variety of experimental approaches, but the available data are inconclusive. For example, ambiguity regarding the presence of a disulfide structure in FGFs was not resolved until the availability of x-ray crystal structure data. Furthermore, the functionally important heparin- and receptor-binding domains have been poorly characterized, with some interpretations being controversial. In this report, we describe a novel fragment of basic FGF (bFGF) with high biological activity [Ser78,96-bFGF(70-153)]. This fragment was generated by pronase treatment of heparin-bound recombinant Glu3,5Ser78,96-bFGF mutant and is active in vitro at an ED50 of about 100 ng/ml. The structure of the fragment and the manner by which it was generated provide additional insight into important aspects of structure-activity relationships in FGFs. Specifically, we conclude that (a) the cysteines in our bFGF mutant do not form a disulfide bond, (b) the high-affinity heparin binding of bFGF critically depends on an intact 3-dimensional structure of the growth factor rather than on specific heparin-binding sequence domains, and (c) the bFGF sequence between residues 70 and 122 is important for high biological activity.

Fluorospectrometric analysis of heparin interaction with fibroblast growth factors.

The fluorescence emission of a single tryptophan residue present in both FGF-1 and FGF-2 was used as a structural probe to directly assess the interaction of the growth factors with heparin or beta-cyclodextran tetradecasulfate. About 20-25% of the fluorescence of either FGF-1 or FGF-2 is quenchable, and is dependent on sulfation of the ligands. The quenchable fluorescence is associated with about 20% of total FGF, suggesting the presence of two fluorospectrometric forms of the protein. The equilibrium dissociation constants, determined by this method, for heparin or beta-cyclodextrin tetradecasulfate binding to FGF-1 are about 1 nM, whereas the values for FGF-2 are 1 and 23 nM, respectively. The method provides a direct tool to evaluate FGF-ligand interaction and assess the structural integrity of the proteins.

Anti-viral activity of human recombinant heparin-binding proteins HBNF and MK

Herpes simplex viruses bind to cell surface heparan sulfate proteoglycans, as a first step of viral infection. We report here that two recombinant heparin-binding proteins HBNF and MK inhibit infectivity of human herpes simplex viruses types 1 and 2 and human cytomegalovirus. Carboxymethylated HBNF and MK, which retain affinity for heparin-Sepharose, do not exhibit anti-viral activities. Arguments are presented that anti-viral effects of HBNF and MK are due to the competition for the specific binding to the cell surface heparan sulfate proteoglycans.

Preliminary X-ray crystallographic analysis of a modified basic fibroblast growth factor

Human basic fibroblast growth factor (hbFGF) has been modified, with Ala3 and Ser5 substituted by glutamic acid, and the purified recombinant protein has been crystallized. The crystals are triclinic (space group P1) with unit cell parameters a = 31.0 A, b = 33.6 A, c = 34.7 A, alpha = 88 degrees, beta = 85 degrees, gamma = 76 degrees, and they diffract to at least 2 A.