Kobielak Agnieszka, Zakrzewska Małgorzata, Kostas Michał, Jakimowicz Piotr, Otlewski Jacek, Krowarsch Daniel
Department of Protein Biotechnology, Faculty of Biotechnology, University of Wroclaw; ul. Joliot- Curie 14 a, 50-383 Wroclaw, Poland.
Protein Pept Lett. 2014 May;21(5):434-43. doi: 10.2174/0929866520666131203102315.
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.
人酸性成纤维细胞生长因子（FGF1）在血管生成和伤口愈合过程中发挥着不可否认的作用，但其在体内稳定性低、半衰期短，这使得人们对其治疗潜力产生了质疑。我们之前的研究表明，通过提高FGF1与改善整体热稳定性直接相关的抗蛋白水解能力，可以实现其延长的生物活性。在本研究中，我们应用了一种替代方法，通过使生长因子对蛋白酶作用高度敏感的片段刚性化来生成持久的FGF1变体。为了确定最容易发生酶促降解的区域，我们使用胰蛋白酶进行有限蛋白水解并结合质谱分析。我们发现，初始蛋白水解切割主要发生在野生型蛋白的C末端区域，这表明其在生长因子降解中起重要作用。基于生物信息学分析，我们在β链XI中引入了两个单突变（C117P、K118V）并将它们组合成一个双突变体。我们测定了所得变体的抗蛋白水解能力、生物物理性质和生物活性。与野生型蛋白相比，所有变体对胰蛋白酶（高达100倍）和胰凝乳蛋白酶降解的敏感性均显著降低。有趣的是，所有变体的热稳定性均不比野生型FGF1高。我们将这种对蛋白水解抗性的显著增加归因于C末端区域的熵稳定作用。
