三疣梭子蟹两种含不同关键氨基酸基序C型凝集素的糖识别机制研究

C-type lectins are a superfamily of pattern recognition receptors (PRRs), which play an important role in the pathogen recognition and activation of the downstream immune response in invertebrate innate immunity. In the present project, Portunus trituberculatus of great scientific significance and economic value is selected as the research object. In previous study, two typical single-CRD containing C-type lectins (PtCTL-2 and PtCTL-3) with different key motifs were identified to have broad nonself recognition activity and display novel carbohydrate binding specificity, in which PtCTL-2 with EPR/GAS motif possessed broad carbohydrate-binding spectrum and the carbohydrate-binding specificity of PtCTL-3 with QPD/WND motif was different from the CRDs in vertebrates. With X-ray diffraction technique, the structure of two C-type lectins including the recombinant form and native form will be resolved. In addition, high-throughput glycan microarrays and isothermal titration microcalorimetric (ITC) technique will be adopted to elucidate their carbohydrate-binding specificity and affinity, and the motif-dependent mechanism. The project will help to illuminate the immune recognition mechanism of Portunus trituberculatus, enrich and develop the research contents of invertebrate immunology, and provide promising ideas for the development of immunological control drugs.

C型凝集素作为一类模式识别受体，能够识别外界病原体，并激活下游免疫应答反应，对缺乏获得性免疫系统的无脊椎动物具有重要的免疫学意义。本项目选取科学意义与经济价值并重的三疣梭子蟹，针对其两种具备广谱“非己”识别活性的典型单结构域C型凝集素（PtCTL-2和PtCTL-3）开展免疫识别机制研究。前期研究发现PtCTL-2的钙离子结合位点2存在新型关键氨基酸基序EPR/GAS且具备广泛的糖结合活性，而具有经典QPD/WND氨基酸基序的PtCTL-3却呈现不同于脊椎动物的糖结合模式。本项目将借助X射线衍射技术解析这两个C型凝集素的重组蛋白及体内天然蛋白的晶体结构，辅以高通量糖芯片及等温滴定微量量热技术阐明关键氨基酸基序的配体结合特异性决定机制。该项目有助于破解三疣梭子蟹的免疫识别机制，丰富和发展无脊椎动物免疫学的研究内容，并为免疫防治药物的研制提供新切入点。

C 型凝集素是海洋无脊椎动物固有免疫系统中的重要模式识别受体（Pattern-recognition receptor, PRR），能识别病原微生物，并激活下游免疫应答反应。本项目综合利用分子生物学、免疫学及结构生物学等技术，以三疣梭子蟹为材料，对典型单结构域C型凝集素PtCTL-2、PtCTL-3开展了免疫识别机制的研究。研究发现PtCTL-2，-3能作为模式识别蛋白识别并结合PAMPs及微生物，凝集不同微生物，还能作为调理素促进血淋巴细胞对微生物的吞噬作用，在三疣梭子蟹免疫防御过程中发挥重要作用。对其识别机制进行探索发现含EPR/GAS的PtCTL-2能特异性结合D-半乳糖，N-乙酰氨基葡糖胺，D-岩藻糖， D-乳糖和 L-岩藻糖，而含 QPD/WND经典关键氨基酸基序的PtCTL-3 的单糖结合特异性也不同于脊椎动物C型凝集素。在对含有新型关键氨基酸基序DPY/WTD的PtCTL-6的研究中发现该分子能够参与对PAMPs及微生物的免疫识别过程；且该分子具备新型糖配体识别模式，能与D-甘露糖和D-岩藻糖特异性结合。研究成果为深入了解海洋无脊椎动物免疫识别分子的结构与功能，阐明海洋无脊椎动物的免疫防御机制奠定了基础，并为免疫防治药物的研制提供新切入点。