Characterization of a cysteine-free analog of recombinant human basic fibroblast growth factor

Arakawa, Tsutomu; Hsu, Yueh-Rong; Schiffer, Steffen; Tsai, Larry B.; Curless, Craig; Fox, Gary M.

doi:10.1016/0006-291x(89)91601-x

Cited by 38 publications

(12 citation statements)

References 6 publications

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“…In fact, as previously shown, the only oligomerization of these CL-dp12⅐bFGF monomer conjugates observed during this study, was the result of disulfide bonding. It seems plausible to discount this as a method for presenting dimeric conjugates to the receptor, as previous experimental data have shown that disulfide bonding of bFGF is not necessary for the growth factor's biological activity (55)(56)(57). In addition, we found that the formation of bFGF disulfide dimers was not influenced by the presence of HS oligosaccharides.…”

Section: Fig 5 [mentioning

confidence: 46%

Monomer Complexes of Basic Fibroblast Growth Factor and Heparan Sulfate Oligosaccharides Are the Minimal Functional Unit for Cell Activation

Pye

Gallagher

1999

Journal of Biological Chemistry

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The interaction of basic fibroblast growth factor (bFGF) with heparan sulfate (HS)/heparin has been shown to strongly enhance the activity of the growth factor although the mechanism of activation is unclear. We have addressed the issue of the minimal stoichiometry of an active HS oligosaccharide⅐bFGF complex by chemically cross-linking the two components to form novel covalent conjugates. The cross-linking procedure produced both monomeric and dimeric bFGF⅐oligosaccharide complexes, which were purified to homogeneity. Dimer conjugates were shown to have been formed as a result of disulfide bridging of monomer conjugates. These monomer conjugates were subsequently found to be biologically active in a mitogenesis assay. We therefore conclude that a monomeric bFGF⅐oligosaccharide complex is the minimal functional unit required for mitogenic stimulation.

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Section: Fig 5 [mentioning

confidence: 46%

Monomer Complexes of Basic Fibroblast Growth Factor and Heparan Sulfate Oligosaccharides Are the Minimal Functional Unit for Cell Activation

Pye

Gallagher

1999

Journal of Biological Chemistry

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“…The formation of intermolecular disulfide bridges involving residues 25 and 92 seems unlikely on steric grounds. All four cysteine residues can be replaced by serine residues without loss of biological activity (30,31), although replacement of Cys-25 and -92 reduces the affinity of bFGF for heparin (30), suggesting that minor structural perturbations may result from the substitution of these buried cysteines.…”

Section: Expression and Structure Determinationmentioning

confidence: 99%

Three-dimensional structure of human basic fibroblast growth factor, a structural homolog of interleukin 1 beta.

Zhang

Cousens

Barr

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

231

160

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The three-dimensional structure of the 146-residue form of human basic fibroblast growth factor (bFGF), expressed as a recombinant protein in yeast, has been determined by x-ray crystallography to a resolution of 1.8 A. bFGF is composed entirely of f-sheet structure, comprising a threefold repeat of a four-stranded antiparallel -meander. The topology of bFGF is Identical to that of interleukin 1fl, showing that although the two proteins share only 10% sequence identity, bFGF, interleukin 1, and their homologs comprise a family of structurally related mitogenic factors. Analysis of the three-dimensional structure in light of functional studies of bFGF suggests that the receptor binding site and the positively charged heparin binding site correspond to adjacent but separate loci on the fl-barrel.

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“…The high similarities between the structures reported here and by Zhu et al (1991) may suggest that Cys substitution makes aFGF more stable by favouring the acquisition of the native structure when the protein refolds during its purification procedure and by eliminating a readily oxidizable group. Arakawa et al (1989) have reported that denaturation and refolding seem to increase considerably the specific activity of a mutant FGF protein in which all the Cys residues have been substituted. Further, aFGF in which Cys residues have been substituted by Ser are considerably more active than the native proteins when they are purified by a protocol that includes a reversed-phase-chromatography step (Seno et al, 1988 ;Ortega et al, 1991), a procedure that involves some refolding of the protein (Katzenstein et al, 1986), but not when the reversed-phase chromatography is avoided (Navarro, M. L., personal communication).…”

Section: Resultsmentioning

confidence: 99%

X‐ray Structure of Native Full‐Length Human Fibroblast‐Growth Factor at 0.25‐nm Resolution

Romero

Pineda‐Lucena

Giménez‐Gallego

1996

European Journal of Biochemistry

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Acidic fibroblast-growth factor (aFGF) is one of the typical members of a group of nine polypeptides of relatively similar amino acid sequence known as the fibroblast-growth-factor family of proteins. Widely distributed throughout the organism, fibroblast-growth factors seem to be involved in numerous physiological processes ranging from control of cell proliferation and differentiation to modulation of animal behaviour and arterial blood pressure. This wide assortment of biological activities explains their involvement in numerous pathologies. Instability and low yields of the purified protein have precluded highresolution structural studies of the physiological form of aFGF. Nevertheless, modifications introduced recently into the synthesis and purification procedures of this protein have allowed preparations of samples that, as shown here, are reliable substrates to obtain crystals suitable for X-ray-diffraction studies. These analyses have allowed us to elucidate the three-dimensional structure of the physiological form of human aFGF by molecular-replacement methods, from the previously reported structure of a shortened form of bovine aFGF that was stabilized by point-directed mutagenesis. The structure was refined at a resolution of 0.25 nm to an R factor of 20.4% for 13 109 reflections between 0.6 nm and 0.25 nm, with rmsd of 1.1 pm and 1.9' from ideal bond lengths and bond angles, respectively. Human aFGF folds according to a ,&trefoil topology. This fold consist of six P-strand pairs. Three of them form a six-stranded P-barrel structure that is capped at one end by the other three pairs arranged in a triangular array. The Nterminus of aFGF up to residue Pro19 appears flexible in the structure and does not specifically interact with the rest of the molecule.

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Characterization of a cysteine-free analog of recombinant human basic fibroblast growth factor

Cited by 38 publications

References 6 publications

Monomer Complexes of Basic Fibroblast Growth Factor and Heparan Sulfate Oligosaccharides Are the Minimal Functional Unit for Cell Activation

Monomer Complexes of Basic Fibroblast Growth Factor and Heparan Sulfate Oligosaccharides Are the Minimal Functional Unit for Cell Activation

Three-dimensional structure of human basic fibroblast growth factor, a structural homolog of interleukin 1 beta.

X‐ray Structure of Native Full‐Length Human Fibroblast‐Growth Factor at 0.25‐nm Resolution

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