硫酸乙酰肝素寡糖的高效合成及与RAGE相互作用研究

Heparan sulfate (HS) is indispensable as active substance on the cell surface and extracellular matrix, which plays important biological roles in cell differentiation and proliferation, cell migration, immune regulation, etc. Although HS possesses diverse biological activities, it is difficult to obtain the oligosaccharides with precise structure due to their complexity which limits the interaction study between these molecules and functional proteins. Receptor for advanced glycation end-products (RAGE) is involved in diverse signal transductions with multiple proteins, however HS oligosaccharides can regulate those signals through the oligomerization of RAGE. This proposal plans to develop novel fluorous tags tolerated by various HS synthetic enzymes and released selectively, and combine the advantages of chemoenzymatic synthesis and fluorous solid phase extraction (FSPE) to achieve the efficient preparation of HS oligosaccharides with distinct structures. The patterns of HS oligosaccharides-RAGE interactions are determined by glycochips, SPR and multiangle laser-light scattering (MALLS), and their qualitative and quantitative analysis and kinetic constants are obtained subsequently. The structure characteristics of HS oligosaccharides are elucidated in the degree of polymerization, sites of sulfate groups and monosaccharides configuration for regulating RAGE, which may supply the scientific basis for understanding the mechanism between HS-RAGE complexes and key proteins for the regulation of signal transductions.

硫酸乙酰肝素（HS）是细胞表面和外基质不可缺少的活性物质，对细胞分化增殖、细胞迁移及免疫调节等具有重要的生物学作用。虽然HS活性广泛，但其细微结构复杂多样，很难有效获得结构明确的寡糖，从而限制了该类分子与功能蛋白相互作用研究。高级糖基化终产物受体（RAGE）参与多种关键蛋白的信号传导，而HS寡糖通过介导RAGE形成寡聚体实现对信号传导的调控。本项目拟构建能够被HS合成酶耐受且可以选择性脱除的氟标签，结合化学酶法合成与氟固相萃取（FSPE）的优势，实现系列结构明确的HS寡糖的高效制备。利用糖芯片、表面等离子体共振及多角度激光散射技术，对合成的HS寡糖与RAGE的作用模式进行定性、定量分析并获得其动态结合参数，从糖链聚合度、硫酸化位点及单糖构型等方面归纳出HS寡糖调控RAGE聚合的结构特征，为揭示HS-RAGE体系参与蛋白间信号传导的机制提供科学依据。

硫酸乙酰肝素（Heparan sulfate, HS）分布在细胞膜表面和细胞外基质中，通过与多种功能蛋白的特异性结合，影响生物体内细胞间的信号转导、调控等重要生理过程。天然来源获取的硫酸乙酰肝素资源有限，且结构具有不均一性，限制其深入构效关系研究。本项目利用化学酶法合成了系列硫酸乙酰肝素寡糖化合物和糖聚合物。通过PmHS2介导的酶法糖基化反应和化学区域选择性硫酸化，高效获得系列硫酸乙酰肝素寡糖化合物；通过自由基聚合（FRP）与化学酶法合成联用技术，制备侧链长度可控和不同硫酸化修饰的结构明确硫酸乙酰肝素寡糖聚合物。利用表面等离子体共振（SPR）作用仪，实现了硫酸乙酰肝素类寡糖聚合物与RAGE蛋白之间相互作用模式评价，研究发现硫酸乙酰肝素三糖聚合物，具有最强的RAGE蛋白结合活性。综合来讲，寡糖链长度和硫酸化形式对糖聚合物与功能蛋白的结合产生重要影响，硫酸乙酰肝素三糖聚合物有望未来开发成高效的RAGE蛋白拮抗剂。