基于AFM单分子力谱的蝉拟青霉多糖超分子结构表征及与TLR4受体靶向识别研究

It is well known that the polysaccharide of Paecilomyces cicadae has potent immunomodulatory activity and it could be stimulated by TLR4 target recognition of macrophages. This kind of biological activity is supposed to be with its certain supramolecular structure, such as chain conformation、mechanical property and dynamics performance. However, few literature has been reported about the mechanism of TLR4 target recognition of FPCPS polysaccharide. In this project, we would combine the theory of polymer solution、single molecule force spectroscopy and statistics to analyze the structure-immunomudulatory activity with the help of multipe laser light scattering (MLLS) and single molecule force spectroscopy (SMFS). The conformation of FPCPS in different solution would be determined by MLLS and AFM. When the AFM tip is continuously approached to and retracted from the biological sample, it monitors the interaction force. Based on the obtained force–extension curves,SMFS would provide key insight into the molecular elasticity and localization of FPCPS single molecule, either on isolated system or on cellular surface. LPS (used as comparison) and FPCPS would be modified before the SMFS testing. Then, the relationship of chain conformation properties and TLR4 target recognition would be elucidated based on the analysis of danamic force spectroscopy of two complexes (LPS-CD14 and FPCPS-CD14). After the clear understanding of TLR4 recognition on cell face, it may provide better foundation of the immunomodulatory food for FPCPS functional application.

食用菌蝉拟青霉多糖具有较强的免疫调节活性，文献研究表明，其胞内多糖可通过激活TLR4细胞信号通路达到免疫增强的作用，但关于该多糖如何与细胞膜上的靶向分子相互作用激活TLR4下游通路尚未见报道，其活性由它在溶液中的超分子构象决定。本项目拟在蝉拟青霉多糖已有的研究基础上，将现代高分子溶液理论、单分子力谱及统计学相结合，用动静态激光光散射研究FPCPS多糖在不同溶液中的超分子构象变化，用单分子力谱测量其超分子单链弹性性质，同时以LPS脂多糖为对照，巧妙地将FPCPS与LPS分别修饰在探针上，将CD14靶向分子修饰在基底，通过研究FPCPS-CD14、LPS-CD14两对复合物的最可断裂力及动态力学谱，获得它们的结合/解离性质及作用模式，从而判断FPCPS与CD14靶向分子的作用机理，为进一步在单分子水平上揭示该多糖的免疫活性及新功能食品开发提供重要理论基础。

食用菌蝉拟青霉(Paecilomyces cicadae Miq. Samson)多糖具有较强的免疫调节作用，可通过激活TLR4细胞信号通路达到免疫增强的作用，但关于该多糖如何与细胞膜上的靶向分子相互作用激活TLR4下游通路尚未见报道，其活性由它在溶液中的超分子构象决定。项目组通过本课题的研究，获得了新资源食品蝉拟青霉活性多糖FPCPS的单分子弹性拉伸技术，单分子针尖修饰技术，单分子与单分子互作的动态力学技术，积累了具有一定空间构象的多糖与细胞靶向分子相互结合的丰富经验。将现代高分子理论与力学相结合，使用激光光散射仪、原子力显微镜等一系列手段研究了蝉拟青霉FPCPS多糖分子高级构象转变过程，获得了其单分子弹性特征，构建了该多糖的MFJC力学模型，获得了FPCPS/CD14、LPS/CD14两对单分子相互作用力，并揭示了它们同时相互结合与解离的动态性质，初步阐明FPCPS作用于免疫细胞TLR4受体的作用机理。研究发现，FPCPS在纯水中以单链棒状存在，单分子形貌参数为：Lc为453±65 nm，Ree为316±25 nm，Lp为0.82±0.03 nm，Height为0.67±0.05 nm；通过单分子力学拉伸获得其M-FJC模型参数：Lcontour轮廓长度425 ±12 nm，lkuhn库恩长度0.65±0.08 nm，它的链弹性常数Ksegment为66.5±0.3 nN/nm，其K0值为43.2 nN；探知单对FPCPS-CD14相互作用力为129± 7 pN，而单对LPS-CD14相互作用力为174±15 pN；利用动力学谱揭示两对复合物相互作用的势垒信息，跨跃LPS-CD14两者结合所需的能垒距离Xβ为0.19±0.03 nm，解离速率常数Koff为0.26±0.09 s-1，平均寿命τ为3.8±0.11 s；跨跃FPCPS-CD14两者结合所需的能垒距离Xβ为0.28±0.08 nm，解离速率常数Koff为0.37±0.12 s-1，平均寿命τ为2.7±0.16 s。由此可以进一步得知FPCPS与免疫细胞上的靶向分子CD14相结合，但作用能力与LPS比较起来相对较弱，但仍可以发挥一定的免疫调节作用。