Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application

Abstract

Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and 3 buried cysteine (Cys) residues (at positions 16, 83, and 117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all 3 positions without also introducing further substantial instability. The mutational introduction of a novel Cys residue (Ala66Cys) that forms a stabilizing disulfide bond (i.e., cystine) with one of the extant Cys residues (Cys83) effectively eliminates one Cys while increasing overall stability. This increase in stability offsets the associated instability of remaining Cys substitution mutations and permits production of a Cys-free form of FGF-1 (Cys16Ser/Ala66Cys/Cys117Ala) with only minor overall instability. The addition of a further stabilizing mutation (Pro134Ala) creates a Cys-free FGF-1 mutant with essentially wild-type biophysical properties. The elimination of buried free thiols in FGF-1 can substantially increase the protein half-life in cell culture. Here, we show that the effective cell survival/mitogenic functional activity of a fully Cys-free form is also substantially increased and is equivalent to wild-type FGF-1 formulated in the presence of heparin sulfate as a stabilizing agent. The results identify this Cys-free FGF-1 mutant as an advantageous “second generation” form of FGF-1 for therapeutic application.

Proliferation of Human Corneal Endothelia in Organ Culture Stimulated by Wounding and the Engineered Human Fibroblast Growth Factor 1 Derivative TTHX1114

“…TTHX1114 (human N-methionyl-FGF1, 141 amino acid form, containing the substitutions Cys16Ser, Ala66Cys, and Cys117Val using the 140 aa numbering system (see Eveleth et al 37 and Xia et al 39 ) was prepared by the Frederick National Laboratory for Cancer Research, Biopharmaceutical Development Program.…”

“…The latter was tested using TTHX1114, an engineered version of human FGF1 that has been shown to stimulate rabbit corneal epithelial cells and protect them from chemical damage, 37 and is a close relative of FGFs that accelerate dermal wound healing in diabetic animals. 38,39 The utility of FGFs is limited by their instability and short half-life in biological systems, consistent with their natural role as signals intended to be limited in time and space. TTHX1114 incorporates several amino acid substitutions to the FGF1 protein known to make the molecule more stable in biological systems.…”

Functionalization of a symmetric protein scaffold: Redundant folding nuclei and alternative oligomeric folding pathways

“…This Cys is a free reactive thiol that can cause unwanted dimer formation, and an Ala substitution has been reported as the most stable substitution at this position. 42 These substitutions were modeled into the 19-mer region of residues 112-130 of the FGF-1 crystal structure (RCSB accession 1JQZ) to generate a putative HS-binding peptide cassette. This modified FGF-1 structure was then overlaid onto the symmetric designed β-trefoil protein Symfoil-4T (RCSB accession 3O4B) using the following regions of backbone atoms: 12-16, 19-47, 54-67, 70-76, 81-90, 94-102, 107-112, 129-136, yielding a 0.63 Å rmsd for the overlaid atoms.…”

Unraveling the Connection between Fibroblast Growth Factor and Bone Morphogenetic Protein Signaling

“…FGF ligands also experience short half-lifes, due to free cysteines. Consequently, not every cell capable of FGF signaling can respond to every ligand, and signaling becomes cell-type specific [ 1 , 13 , 14 ]. Receptor internalization is then able to limit the signal duration [ 2 ], at least if the receptor is subsequently degraded.…”