Structural determinants of heparin–transforming growth factor-β1 interactions and their effects on signalling

Transforming growth factor-ß1 (TGF-ß1, Uniprot: P01137) is a heparin-binding protein that has been implicated in a number of physiological processes, including the initiation of chondrogenesis by human mesenchymal stem cells (hMSCs). Here, we identify the molecular features in the protein and in heparin required for binding and their effects on the potentiation of TGF-ß1’s activity on hMSCs. Using a proteomics “Protect and Label” approach, lysines K291, K304, K309, K315, K338, K373, K375 and K388 were identified as being directly involved in binding heparin. Competition assays in an optical biosensor demonstrated that TGF-ß1 does require N- and 6-O-sulfate groups for binding but that 2-O-sulfate groups are unlikely to underpin the interaction. Heparin-derived oligosaccharides as short as degree of polymerization (dp) 4 have a weak ability to compete for TGF-ß1 binding to heparin, which increases with the length of the oligosaccharide to reach a maximum between dp18 and dp24. In cell-based assays, heparin, 2-O-, 6-O- and N-desulfated re-N-acetylated heparin and oligosaccharides 14–24 saccharides (dp14–24) in length all increased the phosphorylation of SMAD2 after 6 h of stimulation with TGF-ß1. The results provide the structural basis for a model of heparin/heparan sulfate binding to TGF-ß1 and demonstrate that the features in the polysaccharide required for binding are not identical to those required for sustaining the signaling by TGF-ß1 in hMSCs.转化生长因子-β1（"Transforming growth factor-β1"，TGF-β1，UniProt编号：P01137）是一种肝素结合蛋白，已被证实参与诸多生理过程，其中包括人间充质干细胞（"human mesenchymal stem cells"，hMSCs）的软骨发生启动。本研究明确了该蛋白与肝素结合所需的分子特征，以及二者结合对TGF-β1在hMSCs中活性增强的影响。本研究采用蛋白质组学"Protect and Label"技术，鉴定出赖氨酸残基K291、K304、K309、K315、K338、K373、K375与K388直接参与肝素结合过程。光学生物传感器竞争实验结果显示，TGF-β1结合肝素确实需要N-和6-O-硫酸基团，但2-O-硫酸基团并非介导该相互作用的关键基团。肝素来源的寡糖即便聚合度（"degree of polymerization"，dp）低至4，也具备微弱的竞争TGF-β1与肝素结合的能力，且该竞争活性随寡糖链长增加而逐步增强，在聚合度18至24时达到峰值。细胞水平实验结果显示，肝素、2-O-、6-O-及N-脱硫酸化再N-乙酰化肝素，以及链长为14~24个糖单元（dp14~24）的寡糖，均可在TGF-β1刺激6小时后提升SMAD2的磷酸化水平。本研究明确了肝素/硫酸乙酰肝素（"heparan sulfate"）结合TGF-β1的结构模型基础，并证实多糖分子中参与结合TGF-β1的结构特征，与维持TGF-β1在hMSCs中信号传导所需的结构特征并不一致。