The purification of reduced β2-glycoprotein I showed its native activity in vitro.
The purification of reduced β2-glycoprotein I showed its native activity in vitro.
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还原型 β2-糖蛋白 I 的纯化显示其体外天然活性
DOI：
10.1186/s12944-017-0555-x
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发表时间：
2017-09-13
影响因子：
4.5
通讯作者：
Yu P
中科院分区：
文献类型：
--
作者：
Zhou S;Lu M;Zhao J;Liu S;Li X;Zhang R;Liu H;Yu P
新的证据表明，还原型β2-糖蛋白I（β2GPI）具有抗氧化应激和抗炎活性。然而，细节仍然知之甚少。本研究旨在制备具有天然生物活性的稳定还原型β2GPI。用高氯酸沉淀法从血浆中分离纯化人β2GPI，然后用SephadexG-25柱层析、SP-HP、AF-heparin HC-650 M和SephacrylS-200柱层析等一系列层析方法进行纯化。用DL-二硫苏糖醇（DTT）活化的硫氧还蛋白-1（TRX-1）还原纯化的人β2GPI。选择谷胱甘肽（GSH）封闭还原型β2GPI中的游离巯基。LC/MS用于验证游离硫醇的位置。Western blot检测β 2 GPI免疫反应性。采用MTS和流式细胞术研究其对氧化应激诱导的人脐静脉内皮细胞（HUVECs）死亡的生物学效应。定量小鼠血清中肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、白细胞介素-10（IL-10）、白细胞介素-12 P70（IL-12 P70）、干扰素-γ（IFN-γ）和单核细胞趋化蛋白-1（MCP-1）的水平，以评估其在脂多糖（LPS）介导的全身性炎症中的抗炎活性。从200 ml血浆中获得约10 mg β2GPI（纯度98.7%）。蛋白质产量为0.05 mg/ml血浆。在体外用TRX-1/DTT还原β2GPI，检测到β2GPI结构域V的Cys 288和Cys 326上的游离巯基。GSH阻断剂在体外稳定了β2GPI。这种降低的β2GPI可以被抗β2GPI抗体识别，可以显著减少H2 O2处理后HUVECs的死亡，并且可以显著降低LPS刺激后小鼠中TNF-α、IL-6、IFN-γ和MCP-1的水平。通过TRX-1脱氧后用GSH封闭巯基，可在体外获得稳定的还原型β2GPI。这种还原的β2GPI保持与氧化的β2GPI相同的免疫活性，并且具有对抗HUVECs中由H2 O2诱导的氧化应激和LPS介导的小鼠炎症中的炎症的能力。
New evidence has shown that reduced β2-glycoprotein I (β2GPI) has anti-oxidative stress and anti-inflammatory activity. However, the details are still poorly understood. This study aims to prepare stable reduced β2GPI with its native bioactivity in vitro. Human β2GPI was purified from plasma first with perchloric acid precipitation and then purified with a series of chromatography methods including Sephadex G-25 desalting, SP HP, AF-heparin HC-650 M, and Sephacryl S-200. The purified human β2GPI was reduced with thioredoxin-1 (TRX-1) activated by DL-dithiothreitol (DTT). Glutathione (GSH) was selected to block the free thiols in reduced β2GPI. LC/MS was used to verify the location of free thiols. Western blot analysis was used to detect β2GPI immunoreactivity. MTS and flow cytometry were conducted to investigate its biological effect on oxidative-stress-induced death of human umbilical vein endothelial cells (HUVECs). The levels of tumour necrosis factor-alpha (TNF-α),interleukin-6 (IL-6) interleukin-10 (IL-10),interleukin-12P70 (IL-12P70),interferon-gamma (IFN-γ) and monocyte chemoattractant protein −1(MCP-1) in mouse serum were quantified to assess its anti-inflammatory activity in lipopolysaccharide (LPS)-mediated systemic inflammation. We obtained approximately 10 mg β2GPI (purity 98.7%) from 200 ml plasma. The protein yield was 0.05 mg/ml plasma. β2GPI was then reduced by TRX-1/DTT in vitro; the free thiols were detected on Cys288 and Cys326 in domain V of β2GPI. The GSH blockage stabilized the reduced β2GPI in vitro. This reduced β2GPI can be recognized by the anti-β2GPI antibody, can significantly reduce the death of HUVECs after H2O2 treatment and can significantly decrease the levels of TNF-α, IL-6,IFN-γ and MCP-1 in mice upon LPS stimulation. Stable reduced β2GPI can be obtained in vitro by TRX-1 deoxidation followed by the blockage of thiols with GSH. This reduced β2GPI maintains the same immunological activity as oxidized β2GPI and has the ability to counter the oxidative stress induced by H2O2 in HUVECs and inflammation in LPS-mediated inflammation in mice.
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作者：
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DOI：
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发表时间：
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影响因子：
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作者：
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通讯作者：
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