Human cell-expressed tag-free rhMFG-E8 as an effective radiation mitigator.
Human cell-expressed tag-free rhMFG-E8 as an effective radiation mitigator.
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人细胞表达无标签的重组人MFG-E8作为一种有效的辐射缓释剂。
DOI：
10.1038/s41598-023-49499-y
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发表时间：
2023-12-13
影响因子：
4.6
通讯作者：
中科院分区：
文献类型：
--
作者：
人乳脂肪小球表皮生长因子(MFG-E8)作为一种桥梁分子，促进专业吞噬细胞对死亡细胞的清除。大肠杆菌表达的组氨酸标记的重组人MFG-E8(rhMFG-E8)在各种疾病条件下都具有保护作用。然而，由于重组蛋白糖基化、错误折叠和抗原性的可能性，大肠杆菌表达的组氨酸标记的重组人MFG-E8不适合人类治疗。因此，我们推测，人细胞表达的、无标签的重组人MFG-E8将具有合适的结构和功能，有望成为治疗包括辐射损伤在内的炎症性疾病的安全有效的新型生物材料。我们将人MFG-E8全长编码序列克隆到哺乳动物载体中，并在HEK293细胞中表达，获得了一种新的无标签的重组人MFG-E8蛋白。该构建物含有胱抑素S的前导序列，以最大限度地将重组人MFG-E8分泌到培养基中。在蛋白纯化和鉴定后，我们首先对其体外生物学活性进行了评价。然后，我们利用一种实验性的辐射损伤啮齿动物模型，即局部全身照射(PBI)，在体内测定了它的有效性。HEK293细胞培养上清液经浓缩、纯化后，以标准的人MFG-E8为对照，经SDS-PAGE鉴定，并用吉祥物进行多肽质量指纹图谱分析。人细胞表达的无标签的重组人MFG-E8的生物学活性优于大肠杆菌表达的组氨酸标签的重组人MFG-E8。毒性、稳定性和药代动力学研究表明，无标签的重组人MFG-E8是安全的，经冷冻干燥和长期储存后高度稳定，循环中的最终消除半衰期至少为1.45h。在15GyPBI模型中，无标签的rhMFG-E8治疗后30天存活率呈剂量依赖性提高，30d存活率为89%，显著高于赋形剂组的25%。用概率比特法计算的剂量修正因子为1.058。无标签的rhMFG-E8还可以减轻PBI后的胃肠道损伤，这表明它可能成为治疗辐射损伤的医学对策的候选药物。我们的新的人细胞表达的无标签的rhMFG-E8具有适当的结构和功能特性，将被进一步开发为治疗严重急性辐射损伤受害者的安全有效的治疗方法。
Human milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. E. coli-expressed histidine-tagged recombinant human MFG-E8 (rhMFG-E8) is protective in various disease conditions. However, due to improper recombinant protein glycosylation, misfolding and the possibility of antigenicity, E. coli-expressed histidine-tagged rhMFG-E8 is unsuitable for human therapy. Therefore, we hypothesize that human cell-expressed, tag-free rhMFG-E8 will have suitable structural and functional properties to be developed as a safe and effective novel biologic to treat inflammatory diseases including radiation injury. We produced a new tag-free rhMFG-E8 protein by cloning the human MFG-E8 full-length coding sequence without any fusion tag into a mammalian vector and expressed it in HEK293-derived cells. The construct includes the leader sequence of cystatin S to maximize secretion of rhMFG-E8 into the culture medium. After purification and confirmation of the protein identity, we first evaluated its biological activity in vitro. We then determined its efficacy in vivo utilizing an experimental rodent model of radiation injury, i.e., partial body irradiation (PBI). HEK293 cell supernatant containing tag-free rhMFG-E8 protein was concentrated, purified, and rhMFG-E8 was verified by SDS-PAGE with the standard human MFG-E8 loaded as control and, mass spectrometry followed by analysis using MASCOT for peptide mass fingerprint. The biological activity of human cell-expressed tag-free rhMFG-E8 was superior to that of E. coli-expressed His-tagged rhMFG-E8. Toxicity, stability, and pharmacokinetic studies indicate that tag-free rhMFG-E8 is safe, highly stable after lyophilization and long-term storage, and with a terminal elimination half-life in circulation of at least 1.45 h. In the 15 Gy PBI model, a dose-dependent improvement of the 30-day survival rate was observed after tag-free rhMFG-E8 treatment with a 30-day survival of 89%, which was significantly higher than the 25% survival in the vehicle group. The dose modification factor (DMF) of tag-free rhMFG-E8 calculated using probit analysis was 1.058. Tag-free rhMFG-E8 also attenuated gastrointestinal damage after PBI suggesting it as a potential therapeutic candidate for a medical countermeasure for radiation injury. Our new human cell-expressed tag-free rhMFG-E8 has proper structural and functional properties to be further developed as a safe and effective therapy to treat victims of severe acute radiation injury.
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