Studies on antioxidant system by a new antioxidant protein family

我们发现了一种分子质量为23 kDa的新蛋白，命名为MSP23，它是由氧化应激诱导的小鼠腹腔巨噬细胞，并克隆了该蛋白的cDNA。为了研究MSP23的功能，本研究从肝脏中纯化了MSP23的同源蛋白。结果表明，纯化的MSP23具有硫醇特异的抗氧化活性，并能保护谷氨酰胺合成酶不被金属-硫醇混合氧化失活。还发现MSP23与氯化血红素结合，MSP23的硫醇特异的抗氧化活性因结合而丧失。MSP23在二硫苏糖醇存在下防止了氧化应激导致的酶失活。这意味着硫醇化合物参与了氧化应激所氧化的MSP23的还原。因此，我们在体外研究了在反应混合物中加入硫氧还蛋白时MSP23的抗氧化活性，以探索MPS23的氧化还原系统。然而，硫氧还蛋白系统不能作为MSP23的还原系统。与MSP23相关的氧化还原系统值得进一步探索。我们已经研究了氧化应激在其他类型细胞中诱导MPS23的作用。在培养的血管平滑肌细胞中，氧化低密度脂蛋白强烈诱导MSP23的表达，提示该蛋白的抗氧化活性参与了动脉粥样硬化的发生发展。另一方面，尽管氧化应激与急性胰腺炎的发病机制有关，但在雨蛙素诱导的急性胰腺炎中没有诱导MSP23的表达。由于难以从肝脏中获得足够数量的纯化蛋白，我们试图使用酵母表达系统获得大量的MPS23。将MPS23基因亚克隆到酵母表达载体中，对转化子进行筛选。这些转化子产生了MSP23，但产量远远低于预期。

We have found that a novel protein with a molecular mass of 23 kDa, designated MSP23, is induced by oxidative stress in mouse peritoneal macrophages and cloned the cDNA for the protein. In this study, the homologous protein to MSP23 was pruified from the liver in order to investigate the function of MSP23. It was demonstrated that the purified MSP23 possessed the thiol-specific antioxidant activity and that it protected glutamine synthetase from inactivation by a mixed metal-thiol oxidation. It was also found that MSP23 bound hemin and that the thiol-specific antioxidant activity of MSP23 was lost by the binding.MSP23 prevented the inactivation of the enzyme by oxidative stress in the presence of dithiothreitol. This implies that the thiol-compound is involved in the reduction of MSP23 which had been oxidized by the oxidative stress. We, therefore, investigated the antioxidant activity of MSP23 when thioredoxin is added to the reaction mixture in vitro to explore the redox system for MPS23. However, the thioredoxin system did not work as the reduction system for MSP23. The redox systems related to MSP23 deserves further exploration.We have investigated the induction of MPS23 by oxidative stress in other types of cells. In cultured vascular smooth muscle cells, MSP23 was strongly induced by oxidized low density lipoprotein, suggesting that the antioxidant activity of this protein involved in the pathogenesis and progression of atherosclerosis. On the other hand, MSP23 was not induced in caerulein-induced acute pancreatitis although oxidative stress has been implicated in the pathogenesis of this disease.We tried to obtain a large amount of MPS23 using the yeast expression system because it was difficult to have enough amount of the purified protein from the liver. The cDNA of MPS23 was subcloned into the yeast expression vector and the transformants were screened. These transformants produced MSP23 but the amount was far less than expected.