Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
Ticks Tick Borne Dis. 2021 Mar;12(2):101623. doi: 10.1016/j.ttbdis.2020.101623. Epub 2020 Dec 14.
Babesia microti is a zoonotic pathogen that mainly parasitizes mammalian erythrocytes. Oxidative stress can induce gene mutation, protein denaturation and lipid peroxidation, such as reactive oxygen species (ROS) induced by hypoxic environment and the host immune system. An antioxidase, B. microti thioredoxin reductase (Bmi TrxR), has been identified in B. microti. We used a combination of homology modeling and domain prediction to explore the functional sites of Bmi TrxR and found that TrxR has three domains. Constructed a mutant pool which His-tag were at the N-terminus (TrxR-Nhis, C105-Nhis, C110-Nhis, C105110-Nhis, C547-Nhis, C552-Nhis, C547552-Nhis) and the His tag were at the N- and C-terminus (TrxR-NChis, C547-NChis, C552-NChis, C547552-NChis). The proteins were expressed as His-tagged fusion proteins in Escherichia coli. The His-tag of TrxR C-terminus were affected the reaction with Trx. The inhibitory efficiency of DNCB was decreased for mutant C547, compared with recombinant TrxR, indicating that the action site of DNCB might be cysteine at position 547. These results indicate that the N-terminal active site of Bmi TrxR plays an important role in accepting electrons and promotes electron transfer. The C-terminus His tag of Bmi TrxR affected the electron transfer and the reducing activity of Bmi TrxR. Reduce reactive oxygen produced in oxidative stress was reduced by Bmi TrxR, which is beneficial to Babesia survival. Therefore, reduction site of TrxR may become a potential target for Babesia microti treatment.
微小巴贝斯虫是一种主要寄生于哺乳动物红细胞的动物源性病原体。氧化应激可诱导基因突变、蛋白质变性和脂质过氧化，如缺氧环境和宿主免疫系统引起的活性氧（ROS）。微小巴贝斯虫中已鉴定出一种抗氧化酶，即微小巴贝斯虫硫氧还蛋白还原酶（Bmi TrxR）。我们使用同源建模和结构域预测相结合的方法，探讨了 Bmi TrxR 的功能位点，发现 TrxR 具有三个结构域。构建了一个突变体池，其组氨酸标签位于 N 端（TrxR-Nhis、C105-Nhis、C110-Nhis、C105110-Nhis、C547-Nhis、C552-Nhis、C547552-Nhis）和 N 和 C 端（TrxR-NChis、C547-NChis、C552-NChis、C547552-NChis）。这些蛋白质在大肠杆菌中表达为 His 标签融合蛋白。TrxR C 端的 His 标签影响与 Trx 的反应。与重组 TrxR 相比，突变体 C547 的 DNCB 抑制效率降低，表明 DNCB 的作用位点可能是 547 位的半胱氨酸。这些结果表明，Bmi TrxR 的 N 端活性位点在接受电子和促进电子转移中起重要作用。Bmi TrxR 的 C 端 His 标签影响电子转移和 Bmi TrxR 的还原活性。Bmi TrxR 降低了氧化应激产生的活性氧，有利于巴贝斯虫的存活。因此，TrxR 的还原位点可能成为微小巴贝斯虫治疗的潜在靶点。
