title: Protease resistant variants of FGF1 with prolonged biological activity.
abstract: Therapeutic potential of human acidic fibroblast growth factor (FGF1) resulting from its undeniable role in angiogenesis and wound healing processes is questioned due to its low stability and short half-life in vivo. Our previous studies showed that prolonged biological activity of FGF1 can be achieved by increasing its proteolytic resistance directly linked to improved global thermostability. In this study, we applied an alternative method of generation of long-lasting FGF1 variants by rigidification of the growth factor's segment highly sensitive to proteases action. In order to determine regions the most prone to enzymatic degradation, we used limited proteolysis by trypsin combined with mass spectrometry analysis. We found that the initial proteolytic cleavages occurred mainly within the C-terminal region of the wild-type protein, pointing on its significant role in growth factor degradation. Based on bioinformatic analysis, we introduced two single mutations (C117P, K118V) within β-strand XI and combined them in a double mutant. We determined resistance to proteolysis, biophysical properties and biological activities of obtained variants. All of them occurred to be significantly less susceptible to trypsin (up to 100-fold) and also to chymotrypsin degradation comparing to the wild-type protein. Interestingly, all variants were not more thermostable than the wild-type FGF1. We attributed this dramatic increase in resistance to proteolysis to entropic stabilization of C-terminal region.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Kobielak
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Zakrzewska
authors.forename: Michał
authors.initials: M
authors.lastname: Kostas
authors.forename: Piotr
authors.initials: P
authors.lastname: Jakimowicz
authors.forename: Jacek
authors.initials: J
authors.lastname: Otlewski
authors.affiliation: Department of Protein Biotechnology, Faculty of Biotechnology, University of Wroclaw; ul. Joliot- Curie 14 a, 50-383 Wroclaw, Poland. daniel@protein.pl.
authors.forename: Daniel
authors.initials: D
authors.lastname: Krowarsch
mesh_terms: D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism*; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D017354:Point Mutation; D011489:Protein Denaturation; D055550:Protein Stability; D059748:Proteolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014357:Trypsin / Q000378:metabolism*
chemical_list: D016220:Fibroblast Growth Factor 1; D002918:Chymotrypsin; D014357:Trypsin