Structure-guided design, generation, and biofunction of PEGylated fibroblast growth factor 2 variants for wound healing
Structure-guided design, generation, and biofunction of PEGylated fibroblast growth factor 2 variants for wound healing
复制标题
用于伤口愈合的聚乙二醇化成纤维细胞生长因子 2 变体的结构引导设计、生成和生物功能
作者：
Sun Jian;Wu Jiamin;Jin Hui;Ying Te;Jin Wei;Fan Miaojuan;Zhou Jianhui;Chen Hui;Jin Litai;Zhou Jie
成纤维细胞生长因子2(FGF2)在组织修复等多种生理功能中发挥着重要作用。然而，由于蛋白酶的降解，FGF2在体内的半衰期较短，从而限制了其临床应用。传统的聚乙二醇化反应主要集中在成纤维细胞生长因子2的N-末端α-氨基上。这些修饰没有考虑对蛋白质功能或结构的潜在影响，有时会导致生物活性降低。在本研究中，我们根据FGF2-FGFR-肝素三元复合体的结构，通过基因突变和聚乙二醇化产生了三个聚乙二醇化的FGF2突变体，并研究了这些聚乙二醇化位点对蛋白质稳定性和生物活性的影响。与天然FGF2相比，所有的PEGFGF2偶联物的稳定性都有了显著的提高。聚乙二醇化的结合物在体外比FGF2更有效地促进细胞的增殖、迁移和血管生成，在体内表现出良好的创面愈合活性，使这些结合物成为潜在的创面修复候选药物。基于结构的计算机辅助修饰揭示了蛋白质的详细结构特征，从而可以有效地对蛋白质进行修饰，以提高稳定性和活性。这种结构导向的聚乙二醇化提供了一种更可靠的修饰策略，应该被应用于基于蛋白质的治疗的合理设计。
Fibroblast growth factor 2 (FGF2) plays an important role in multiple physiological functions such as tissue repair. However, FGF2 has a short half-life in vivo due to protease degradation, thus limiting its clinical application. Traditional PEGylation has typically focused on the N-terminal α-amino group of FGF2. These modifications do not consider potential effects on protein function or structure, and sometimes lead to decreased bioactivity. In this study, we generated three PEGylated FGF2 variants based on the structure of the FGF2–FGFR–heparin ternary complex via gene mutation and PEGylation, and investigated the effects of these PEGylated sites on protein stability and bioactivity. Compared with native FGF2, all PEG–FGF2 conjugates exhibited significantly improved stability. Conjugates PEGylated at a site separated from both binding regions more effectively promoted proliferation, migration and angiogenesis than FGF2 in vitro, and exhibited excellent wound healing activity in vivo, making these conjugates potential therapeutic candidates for wound healing. Computer-assisted modification based on structure reveals the detailed structural characteristics of proteins, allowing efficient protein modification for improved stability and activity. This structure-guided PEGylation offers a more reliable modification strategy and should be applied for the rational design of protein-based therapeutics.