Kleemann R, Kapurniotu A, Frank R W, Gessner A, Mischke R, Flieger O, Jüttner S, Brunner H, Bernhagen J
Chair for Interfacial Engineering, University of Stuttgart, Stuttgart, D-70569, Germany.
J Mol Biol. 1998 Jul 3;280(1):85-102. doi: 10.1006/jmbi.1998.1864.
The molecular mechanism of action of macrophage migration inhibitory factor (MIF), a cytokine with a critical role in the immune and inflammatory response, has not yet been identified. Here we report that MIF can function as an enzyme exhibiting thiol-protein oxidoreductase activity. Using a decapeptide fragment of MIF (MF1) spanning the conserved cysteine sequence motif Cys57-Ala-Leu-Cys60 (CALC), Cys-->Ser mutants (C57S MIF, C60S MIF, and C57S/C60S MIF) of human MIF (wtMIF), and alkylated wtMIF, we show that this activity is mediated by the CALC region and is important for the macrophage-activating properties of MIF. Both wtMIF and MF1 were demonstrated to form an intramolecular disulfide bridge. Using two common oxidoreductase assays, MIF was shown to enzymatically catalyze the reduction of insulin and 2-hydroxyethyldisulfide (HED). Examination of wtMIF and the mutants by far-UV circular dichroism spectroscopy (CD) together with denaturation studies showed that substituting or reducing the cysteine residues of CALC led to a reduced conformational stability of MIF but did not significantly change its overall conformation. A functional role for the CALC region was revealed by subjecting the mutants and alkylated wtMIF to the enzymatic assays. Mutant C60S did not have any enzymatic activity while mutant C57S had a reduced activity. Thiol-modified wtMIF that was alkylated under oxidizing conditions was found to have full enzymatic activity, whereas alkylation of wtMIF under reducing conditions completely eliminated MIF-mediated redox activity. Importantly, further physiological relevance of the disulfide motif was obtained by examining the mutants and alkylated MIF in an immunological assay that involved the macrophage-activating properties of MIF. In this test, mutant C60S was essentially inactive and mutant C57S was partly active, indicating together that at least some of the cytokine-like biological activities of MIF are dependent on the presence of cysteine 57 and 60. Again, use of the alkylated MIF species confirmed the role of the cysteine motif for this MIF activity. In conclusion, our results argue (a) that MIF exhibits enzymatic oxidoreductase activity, (b) that this activity is dependent on the presence of the catalytic center that is formed by cysteine residues 57 and 60, and (c) that certain MIF-mediated immune processes are due to the cysteine-mediated redox mechanism.
巨噬细胞移动抑制因子（MIF）是一种在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。在此我们报告，MIF可作为一种具有硫醇 - 蛋白质氧化还原酶活性的酶发挥作用。利用跨越保守半胱氨酸序列基序Cys57 - Ala - Leu - Cys60（CALC）的MIF十肽片段（MF1）、人MIF（野生型MIF，wtMIF）的Cys→Ser突变体（C57S MIF、C60S MIF和C57S/C60S MIF）以及烷基化的wtMIF，我们发现这种活性由CALC区域介导，并且对MIF的巨噬细胞激活特性很重要。wtMIF和MF1均被证明可形成分子内二硫键。通过两种常见的氧化还原酶测定法，显示MIF可酶促催化胰岛素和2 - 羟基乙二硫醚（HED）的还原。利用远紫外圆二色光谱（CD）结合变性研究对wtMIF及其突变体进行检测，结果表明，取代或还原CALC中的半胱氨酸残基会导致MIF构象稳定性降低，但不会显著改变其整体构象。通过对突变体和烷基化的wtMIF进行酶活性测定，揭示了CALC区域的功能作用。突变体C60S没有任何酶活性，而突变体C57S活性降低。发现在氧化条件下烷基化的硫醇修饰wtMIF具有完全的酶活性，而在还原条件下wtMIF的烷基化则完全消除了MIF介导的氧化还原活性。重要的是，通过在涉及MIF巨噬细胞激活特性的免疫测定中检测突变体和烷基化的MIF，获得了二硫键基序的进一步生理相关性。在该试验中，突变体C60S基本无活性，突变体C57S部分有活性，这共同表明MIF至少部分细胞因子样生物活性依赖于半胱氨酸57和60的存在。同样，使用烷基化的MIF物种证实了半胱氨酸基序对该MIF活性的作用。总之，我们的结果表明：（a）MIF具有酶促氧化还原酶活性；（b）这种活性依赖于由半胱氨酸残基57和60形成的催化中心的存在；（c）某些MIF介导的免疫过程归因于半胱氨酸介导的氧化还原机制。
