Abstract
Fibroblast growth factor 2 (FGF2) is a heparin-binding growth factor with broad mitogenic and cell survival activities. Its effector functions are induced upon the formation of 2:2 FGF2:FGFR1 tetrameric complex. To facilitate receptor activation, and therefore, to improve the FGF2 biological properties, we preorganized dimeric ligand by a covalent linkage of two FGF2 molecules. Mutations of the FGF2 WT protein were designed to obtain variants with a single surface-exposed reactive cysteine for the chemical conjugation via maleimide-thiol reaction with bis-functionalized linear PEG linkers. We developed eight FGF2 dimers of defined topology, differing in mutual orientation of individual FGF2 molecules. The engineered proteins remained functional in terms of FGFR downstream signaling activation and were characterized by the increased stability, mitogenic potential and anti-apoptotic activity, as well as induced greater migration responses in normal fibroblasts, as compared to FGF2 monomer. Importantly, biological activity of the dimers was much less dependent on the external heparin administration. Moreover, some dimeric FGF2 variants internalized more efficiently into FGFR overexpressing cancer cells. In summary, in the current work, we showed that preorganization of dimeric FGF2 ligand increased the stability of the growth factor, and therefore, enhanced its biological activity.
Highlights
Fibroblast growth factor 2 (FGF2) is a member of the family of signaling proteins controlling a plethora of cellular processes, such as proliferation, survival, migration, and differentiation [1,2,3]
Application of single cysteine-containing FGF2 mutants allowed for site-specific PEGylation, and generation of homogenous dimers of defined topology
We hypothesized that long flexible linker could enable protein dimerization without disrupting the mutual arrangement of individual FGF2 molecules in the complex with FGFR
Summary
Fibroblast growth factor 2 (FGF2) is a member of the family of signaling proteins controlling a plethora of cellular processes, such as proliferation, survival, migration, and differentiation [1,2,3]. The binding of FGF2 together with HSPG to the extracellular part of FGFR evokes receptor dimerization and autophosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, initiating multiple signal transduction pathways. FGF2 activity depends on a formation of two 1:1:1 FGF–FGFR–HSPG complexes arranged in a symmetrical dimer [2,6,7]. In view of the fact that FGF2 dimerization is essential for the formation of active ligand–receptor complex, the covalent linkage of two FGF2 molecules should improve the assembly of the FGFR signaling platform. Dimerization could function as a general mode for sensing ligand concentration, thereby facilitating activation of the receptor. This makes protein dimerization a tempting research tool to create growth factor therapeutics with superagonist activity
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
