Studies on antioxidant system by a new antioxidant protein family

我们已经发现，一种新的蛋白质分子量为23 kDa，指定MSP 23，诱导小鼠腹腔巨噬细胞的氧化应激和克隆的cDNA的蛋白质。为了研究MSP 23的功能，本研究从肝脏中纯化了MSP 23的同源蛋白。结果表明，纯化的MSP 23具有巯基特异性的抗氧化活性，它保护谷氨酰胺合成酶从混合金属-巯基氧化失活。研究还发现，MSP 23与氯化血红素结合后，其巯基特异性抗氧化活性丧失，在二硫苏糖醇存在下，MSP 23可防止氧化应激导致的酶失活。这意味着硫醇化合物参与了已经被氧化应激氧化的MSP 23的还原。因此，我们研究了当硫氧还蛋白加入到体外反应混合物中时MSP 23的抗氧化活性，以探索MPS 23的氧化还原系统。然而，硫氧还蛋白系统不作为MSP 23的还原系统起作用。与MSP 23相关的氧化还原系统值得进一步探索。我们已经研究了其他类型细胞中氧化应激对MPS 23的诱导作用。在体外培养的血管平滑肌细胞中，氧化低密度脂蛋白对MSP 23的表达有强烈的诱导作用，提示该蛋白的抗氧化活性参与了动脉粥样硬化的发生和发展。另一方面，在雨蛙素诱导的急性胰腺炎中，虽然氧化应激与该疾病的发病机制有关，但MSP 23没有被诱导。我们试图使用酵母表达系统获得大量的MPS 23，因为很难从肝脏中获得足够量的纯化蛋白。将MPS 23的cDNA亚克隆到酵母表达载体中，并筛选转化子。这些转化体产生MSP 23，但量远低于预期。

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.