冷冻电镜揭示长期低温保存的冰冻人血浆中全长补体C3仍可稳定存在
朱 莉，武 迪，武 一*
冷冻电镜揭示长期低温保存的冰冻人血浆中全长补体C3仍可稳定存在
朱 莉，武 迪，武 一*
（1. 教育部细胞活动与逆境适应重点实验室，兰州大学生命科学学院，甘肃 兰州 730000；2. 西安交通大学基础医学院，陕西 西安 710049）
摘 要 补体是血浆中存在的一组重要蛋白质，在机体遭受病原体感染或组织损伤时通过先天免疫途径被激活，发挥抗感染和清除损伤细胞的作用。补体C3是补体系统的核心成分，在三条补体激活途径中均起到至关重要的作用，且在血浆中的含量最为丰富。冰冻人血浆通常由健康志愿者献血后采集，通过迅速冷冻保存制备而成，广泛用于多种医学治疗。然而，关于冰冻人血浆的低温保存条件和时长对补体C3结构完整性的影响尚缺乏准确详细的评估。在本研究中，作者发现，冰冻人血浆在-20℃保存后转为-80℃的条件下保存长达5年，其中的补体C3组分仍能保持完整的结构，且通过冷冻电镜单颗粒重构达到了2.43 Å的分辨率，其中ASN63和ASN917上发生的糖基化修饰亦可被解析。与已解析的C3晶体结构相比，RMSD值差异较大区域在α链末端的C345C结构域，与解析的ISG65-C3复合体冷冻电镜结构相比，C3部分的RMSD值差异较大处仅在C345C末端的一段α螺旋。结果表明，在严格的低温保存条件下，尽管保存时间较长，冰冻人血浆中的关键补体组分C3仍能保持结构完整性和稳定性，从而保障其生物学功能的发挥。由此推测，血浆中其他重要的补体组分在类似保存条件和时长下也可维持其天然构象。这一结论为实验室或工业中由冰冻人血浆提纯制备补体各组分蛋白时，对冰冻血浆的保存条件及期限的设定提供了一定的科学依据和参考。
关键词 冰冻血浆；补体系统；补体C3；冷冻电镜；三维重构
中图分类号： R457.1；Q31；Q336 文献标识码：ADoi:10.3969/j.issn.1000-6281.2025.01.005
Cryo-EM reveals that the full-length complement C3 remains structurally stable in frozen human plasma under long-term low-temperature storage
ZHU Li，WU Di，WU Yi*
(1. MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou Gansu 730000；2. MOE Key Laboratory of Environment and Genes Related to Diseases, School of Basic Medical Sciences, Xi’an Jiaotong University, Xi’an Shaanxi 710049，China)
Abstract Complement proteins are critical plasma components activated via innate immune pathways in response to pathogen infection or tissue damage, playing a pivotal role in immune defense and the clearance of damaged cells. Complement C3, central to all three complement activation pathways, is the most abundant complement protein in plasma. Frozen human plasma, collected from healthy volunteer donors and rapidly frozen, is widely used in various medical treatments. However, detailed evaluations of how low-temperature storage conditions and duration affect the structural integrity of complement C3 in frozen plasma are lacking. In this study, we found that complement C3 in frozen human plasma, stored at -20℃ and then transferred to -80℃ for up to 5 years, retained its structural integrity. Using single-particle cryo-electron microscopy, we determined complement C3 structure at 2.43 Å resolution, and resolved glycosylation modifications occurring at ASN63 and ASN917. Compared to the C3 crystal structure, the regions with the most significant RMSD differences were located in the C345C domain at the C-terminal of the α-chain. When compared to the Cryo-EM structure of ISG65-C3 complex, the most notable RMSD variation of C3 was confined to a short α-helix at the end of the C345C domain. These findings suggest that under stringent low-temperature conditions, complement C3 in long-term stored frozen human plasma can retain its structural integrity and biological function. It is implied that other key complement components in plasma may similarly retain their native structures under such storage conditions. This study provides a basis for determining the appropriate storage conditions and duration for the purification and preparation of complement proteins from frozen human plasma in both laboratory and industrial settings.
Keywords frozen plasma; complement system; complement component 3; Cryo-EM; three-dimensional reconstruction