Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action.


Abstract

Fibroblast growth factor 1 (FGF-1) shows strong angiogenic, osteogenic and tissue-injury repair properties that might be relevant to medical applications. Since FGF-1 is partially unfolded at physiological temperature we decided to increase significantly its conformational stability and test how such an improvement will affect its biological function. Using an homology approach and rational strategy we designed two new single FGF-1 mutations: Q40P and S47I that appeared to be the most strongly stabilizing substitutions among those reported so far, increasing the denaturation temperature by 7.8 deg. C and 9.0 deg. C, respectively. As our goal was to produce highly stable variants of the growth factor, we combined these two mutations with five previously described stabilizing substitutions. The multiple mutants showed denaturation temperatures up to 27 deg. C higher than the wild-type and exhibited full additivity of the mutational effects. All those mutants were biologically competent in several cell culture assays, maintaining typical FGF-1 activities, such as binding to specific cell surface receptors and activation of downstream signaling pathways. Thus, we demonstrate that the low denaturation temperature of wild-type FGF-1 is not related to its fundamental cellular functions, and that FGF-1 action is not affected by its stability. A more detailed analysis of the biological behavior of stable FGF-1 mutants revealed that, compared with the wild-type, their mitogenic properties, as probed by the DNA synthesis assay, were significantly increased in the absence of heparin, and that their half-lives were extensively prolonged. We found that the biological action of the mutants was dictated by their susceptibility to proteases, which strongly correlated with the stability. Mutants which were much more resistant to proteolytic degradation always displayed a significant improvement in the half-life and mitogenesis. Our results show that engineered stable growth factor variants exhibit enhanced and prolonged activity, which can be advantageous in terms of the potential therapeutic applications of FGF-1.

Submission Details

ID: 4j4tndHa

Submitter: Shu-Ching Ou

Submission Date: Sept. 29, 2018, 10:06 a.m.

Version: 1

Publication Details
Zakrzewska M;Krowarsch D;Wiedlocha A;Olsnes S;Otlewski J,J Mol Biol (2005) Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action. PMID:16126225
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1AFC 1993-07-13T00:00:00+0000 2.7 STRUCTURAL STUDIES OF THE BINDING OF THE ANTI-ULCER DRUG SUCROSE OCTASULFATE TO ACIDIC FIBROBLAST GROWTH FACTOR
1BAR 1992-09-29T00:00:00+0000 2.7 THREE-DIMENSIONAL STRUCTURES OF ACIDIC AND BASIC FIBROBLAST GROWTH FACTORS
2J3P 2006-08-22T00:00:00+0000 1.4 crystal structure of rat FGF1 at 1.4 A
2UUS 2007-03-07T00:00:00+0000 2.2 Crystal structure of the rat FGF1-sucrose octasulfate (SOS) complex.
1AXM 1997-10-16T00:00:00+0000 3.0 HEPARIN-LINKED BIOLOGICALLY-ACTIVE DIMER OF FIBROBLAST GROWTH FACTOR
1DJS 1999-12-03T00:00:00+0000 2.4 LIGAND-BINDING PORTION OF FIBROBLAST GROWTH FACTOR RECEPTOR 2 IN COMPLEX WITH FGF1
1DZC 2000-02-24T00:00:00+0000 0 High resolution structure of acidic fibroblast growth factor. Mutant FGF-4-ALA-(24-154), 24 NMR structures
1DZD 2000-02-24T00:00:00+0000 0 High resolution structure of acidic fibroblast growth factor (27-154), 24 NMR structures
1E0O 2000-04-03T00:00:00+0000 2.8 CRYSTAL STRUCTURE OF A TERNARY FGF1-FGFR2-HEPARIN COMPLEX
1EVT 2000-04-20T00:00:00+0000 2.8 CRYSTAL STRUCTURE OF FGF1 IN COMPLEX WITH THE EXTRACELLULAR LIGAND BINDING DOMAIN OF FGF RECEPTOR 1 (FGFR1)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.5 Fibroblast growth factor 1 Q9N1S8 FGF1_CAPCA
90.7 Fibroblast growth factor 1 Q7M303 FGF1_SHEEP
92.1 Fibroblast growth factor 1 P03968 FGF1_BOVIN
97.8 Fibroblast growth factor 1 P20002 FGF1_PIG
96.4 Fibroblast growth factor 1 P61149 FGF1_RAT
96.4 Fibroblast growth factor 1 P61148 FGF1_MOUSE
97.9 Fibroblast growth factor 1 P34004 FGF1_MESAU
100.0 Fibroblast growth factor 1 Q5NVQ3 FGF1_PONAB
100.0 Fibroblast growth factor 1 P05230 FGF1_HUMAN