Kleemann R, Mischke R, Kapurniotu A, Brunner H, Bernhagen J
Laboratory of Biochemistry, University of Stuttgart, Fraunhofer Institute/Fh-IGB, Germany.
FEBS Lett. 1998 Jul 3;430(3):191-6. doi: 10.1016/s0014-5793(98)00654-1.
The molecular mechanism of action of MIF, a cytokine that plays a critical role in the host immune and inflammatory response, has not yet been identified. We recently demonstrated that MIF is an enzyme that exhibits oxidoreductase activity by a cysteine thiol-mediated mechanism. Here we further investigated this function by examining the reduction of insulin disulfides by wild-type human MIF (wtMIF) using various substrates, namely glutathione (GSH), dihydrolipoamide, L-cysteine, beta-mercaptoethanol and dithiothreitol. The activity of wtMIF was compared to that of the relevant cysteine mutants of MIF and to two carboxy-truncated mutants. Only GSH and dihydrolipoamide were found to serve as reductants, whereas the other substrates were not utilized by MIF. Reduction of insulin disulfides by MIF was closely dependent on the presence of the Cys57-Ala-Leu-Cys60 (CALC) motif-forming cysteines C57 and C60, whereas C81 was not involved (activities: 51+/-13%, 14+/-5%, and 70+/-12% of wtMIF, respectively, and 20+/-3% for the double mutant C57S/C60S). Confirming the notion that the activity of MIF was dependent on the CALC motif in the central region of the MIF sequence, the C-terminal deletion mutants MIF(1-105) and MIF(1-110) were found to be fully active. The favored use of GSH and dihydrolipoamide indicated that MIF may be involved in the regulation of cellular redox processes and was supported further by the finding that MIF expression by the cell lines COS-1 and RAW 264.7 was significantly induced upon treatment with the oxidant hydrogen peroxide.
巨噬细胞迁移抑制因子（MIF）是一种在宿主免疫和炎症反应中起关键作用的细胞因子，其分子作用机制尚未明确。我们最近证明，MIF是一种通过半胱氨酸硫醇介导机制表现出氧化还原酶活性的酶。在此，我们通过使用各种底物（即谷胱甘肽（GSH）、二氢硫辛酰胺、L-半胱氨酸、β-巯基乙醇和二硫苏糖醇）检测野生型人MIF（wtMIF）对胰岛素二硫键的还原作用，进一步研究了该功能。将wtMIF的活性与MIF的相关半胱氨酸突变体以及两个羧基截短突变体的活性进行了比较。结果发现，只有GSH和二氢硫辛酰胺可作为还原剂，而其他底物MIF无法利用。MIF对胰岛素二硫键的还原作用紧密依赖于形成Cys57-Ala-Leu-Cys60（CALC）基序的半胱氨酸C57和C60的存在，而C81不参与其中（活性分别为wtMIF的51±13%、14±5%和70±12%，双突变体C57S/C60S的活性为20±3%）。C末端缺失突变体MIF(1-105)和MIF(1-110)具有完全活性，这证实了MIF的活性依赖于MIF序列中部的CALC基序这一观点。对GSH和二氢硫辛酰胺的优先利用表明，MIF可能参与细胞氧化还原过程的调节，氧化剂过氧化氢处理细胞系COS-1和RAW 264.7后MIF表达显著上调这一发现进一步支持了该观点。
