基于Dectin-1受体识别的酵母葡聚糖酶解片段的链结构及构效关系的研究

The frontier researches are focus on the correlation of structure and activity, and we are confronted with the difficulty that discovers the corresponding structure associated with activity. β-(1→3)-glucan from Saccharomyces cerevisiae is as well known as biological response modifiers (BRMs), but it is lack of water-solubility to limit its application and finding of active structure. Therefore, in previous work, a new β-(1→3)-glucanase was used for hydrolyzation of the water-insoluble β-(1→3)-glucan from Saccharomyces cerevisiae to prepare bioactive fractions. In this proposal, the above bioactive fractions are subjected to fractionation further to obtain subfractions with different molecular weight and chain conformation. On the basis of molecular parameters measured by dynamic laser light scattering (D-LLS), size exclusion chromatography combined with LLS, viscometer and refractive index detector (SEC-LLS-Vis-RI), atomic force microscopy (AFM), and transmission electron microscopy (TEM), the wormlike cylinder model and single molecule force spectroscopy (SMFS) are introduced to determine molecular chain conformation of these β-(1→3)-glucan fractions. The screening model based on Dectin-1 recognition capability to β-(1→3)-glucan and murine RAW 264.7 macrophages model are used to discover and confirm the corresponding structure associated with activity in order to clarify the relationship between chain structure and immunomodulation. This work will be not only favorable to the development and application of β-(1→3)-glucan from Saccharomyces cerevisiae in the field of functional food, but also contribute new thought to associating structure (especially chain conformation) with immune activities of β-(1→3)-glucan, due to the screening model based on Dectin-1 recognition to β-(1→3)-glucan.

多糖链结构与活性间的构效关系是阐明和发掘特定多糖结构的热点前沿问题，找寻多糖活性结构域也是当前研究的难点。酵母葡聚糖（SG）是公认的生物应答效应物，然而水溶性差，制约其活性利用及活性结构域的发掘。前期我们利用实验室筛选的β-(1→3)-葡聚糖酶水解啤酒SG获得宽分布的免疫活性片段。本项目拟从上述片段着手，运用D-LLS，SEC-LLS-Vis-RI、NMR、AFM、TEM基础上，引入蠕虫链模型的分子模拟和单分子力谱剖析SG片段的分子尺寸、链构象，建立其分子链构象模型；利用Dectin-1受体对不同链结构SG的特异性识别差异，筛选活性片段，并通过诱导小鼠巨噬细胞的Dectin-1表达及相关细胞因子的分泌来验证，发现并确证免疫活性的特征结构域，阐明链结构与免疫调节活性的构效关系。本研究为揭示营养强化剂SG的活性结构及构效关系提供理论依据；基于特异性受体识别的筛选为活性结构域的发掘提供了新思路。

酵母葡聚糖SG是公认的生物应答效应物，然而其水溶性差，制约其活性的利用及活性结构域的发掘。本项目采用碱提酸解SG，再通过凝胶渗透色谱柱分级，制备了14个不同分子尺寸和链构象的水溶性酵母葡聚糖级分YBG。用SEC-LLS-Vis-RI测得14个YBG的重均分子量Mw在39.36-4146KDa，其Mark-Houwink方程指数α值在0-0.4之间。由分形维数df=3/(1+α)，得df值在2-3。由分子参数ρ=<S2>z1/2/Rh，发现当Mw<229.8KDa时，ρ>2，呈刚性链；当Mw>2836KDa时，ρ<0.77，呈紧缩线团，接近密度均匀球体。引入蠕虫状圆筒模型分子模拟得到YBG的单位围长摩尔质量ML和持续长度q，当Mw=107.6KDa时，ML=2911nm-1，q=1.1nm，呈刚性链；Mw=3705KDa时，ML=8824nm-1，q=1.3nm；随Mw增大，q变化不大，ML急剧增加，即密度显著增加，说明随Mw增加，YBG由刚性伸展的链变为紧缩线团。采用CD、荧光光谱和微量热泳动MST研究YBG与Dectin-1的相互作用，CD结果显示：YBG的添加导致Dectin-1的部分无规卷曲转变为α-螺旋。荧光光谱结果显示：Mw在63.52-3705KDa的YBG皆会使Dectin-1荧光强度减弱，说明它们与Dectin-1间皆存在相互作用；Stern-Volmer曲线和log((F0-F)/F)~log([Q]-n(F0-F)[P]/F0)曲线得出Mw为2832KDa的YBG具有最高的淬灭常数6.11×105M-1和结合常数9.15×105M-1，说明其与Dectin-1的结合能力最强。MST结果显示：YBG与Dectin-1间的解离常数Kd和解离百分比很低，尤其是Mw=2832KDa时，Kd=5.31+/-0.464nM，说明其与Dectin-1的相互作用最强。此外，荧光光谱得到的结合位点n≈1和MST的Thermophoresis图呈S曲线，皆证明YBG与Dectin-1间是1:1结合的。综上，Mw为2832KDa，呈紧缩球形构象的水溶性酵母葡聚糖能被免疫细胞上的Dectin-1识别，并紧密结合，进而激活免疫细胞分泌细胞因子，诱导免疫反应。本项目基于特异性受体(Dectin-1)识别的新思路，发现了酵母葡聚糖的活性特征结构，揭示了链结构与活性的构效关系。