Kleemann R, Kapurniotu A, Mischke R, Held J, Bernhagen J
Laboratory of Biochemistry, University of Stuttgart, Germany.
Eur J Biochem. 1999 May;261(3):753-66. doi: 10.1046/j.1432-1327.1999.00327.x.
Macrophage migration inhibitory factor (MIF) displays both cytokine and enzyme activities, but its molecular mode of action is still unclear. MIF contains three cysteine residues and we showed recently that the conserved Cys57-Ala-Leu-Cys60 (CALC) motif is critical for the oxidoreductase and macrophage-activating activities of MIF. Here we probed further the role of this catalytic centre by expression, purification, and characterization of the cysteine-->serine mutants Cys60Ser, Cys57Ser/Cys60Ser, and Cys81Ser of human MIF and of mutants Ala58Gly/Leu59Pro and Ala58Gly/Leu59His, containing a thioredoxin (Trx)-like and protein disulphide isomerase (PDI)-like dipeptide, respectively. The catalytic centre mutants formed inclusion bodies and the resultant mutant proteins Cys57Ser/Cys60Ser, Ala58Gly/Leu59Pro, and Als58Gly/Leu59His were only soluble in organic solvent or 6 m GdmHCl when reconstituted at concentrations above 1 microgram.mL-1. This made it necessary to devise new purification methods. By contrast, mutant Cys81Ser was soluble. Effects of pH, solvent, and ionic strength conditions on the conformation of the mutants were analysed by far-UV CD spectropolarimetry and mutant stability was examined by denaturant-induced unfolding. The mutants, except for mutant Cys81Ser, showed a close conformational similarity to wild-type (wt) MIF, and stabilization of the mutants was due mainly to acid pH conditions. Intramolecular disulphide bond formation at the CALC region was confirmed by near-UV CD of mutant Cys60Ser. Mutant Cys81Ser was not involved in disulphide bond formation, yet had decreased stability. Analysis in the oxidoreductase and a MIF-specific cytokine assay revealed that only substitution of the active site residues led to inactivation of MIF. Mutant Cys60Ser had no enzyme and markedly reduced cytokine activity, whereas mutant Cys81Ser was active in both tests. The Trx-like variant showed significant enzyme activity but was less active than wtMIF; PDI-like MIF was enzymatically inactive. However, both variants had full cytokine activity. Together with the low but nonzero cytokine activity of mutant Cys60Ser, this indicated that the cytokine activity of MIF may not be tightly regulated by redox effects or that a distinguishable receptor mechanism exists. This study provides evidence for a role of the CALC motif in the oxidoreductase and cytokine activities of MIF, and suggests that Cys81 could mediate conformational effects. Availability and characterization of the mutants should greatly aid in the further elucidation of the mechanism of action of the unusual cytokine MIF.
巨噬细胞移动抑制因子（MIF）兼具细胞因子和酶活性，但其分子作用模式仍不清楚。MIF含有三个半胱氨酸残基，我们最近发现保守的Cys57-Ala-Leu-Cys60（CALC）基序对MIF的氧化还原酶和巨噬细胞激活活性至关重要。在此，我们通过对人MIF的半胱氨酸→丝氨酸突变体Cys60Ser、Cys57Ser/Cys60Ser和Cys81Ser，以及分别含有硫氧还蛋白（Trx）样和蛋白质二硫键异构酶（PDI）样二肽的突变体Ala58Gly/Leu59Pro和Ala58Gly/Leu59His进行表达、纯化和特性分析，进一步探究了这个催化中心的作用。催化中心突变体形成了包涵体，由此产生的突变蛋白Cys57Ser/Cys60Ser、Ala58Gly/Leu59Pro和Als58Gly/Leu59His只有在浓度高于1μg/mL进行重构时才溶于有机溶剂或6m盐酸胍（GdmHCl）。这使得有必要设计新的纯化方法。相比之下，突变体Cys81Ser是可溶的。通过远紫外圆二色光谱偏振法分析了pH、溶剂和离子强度条件对突变体构象的影响，并通过变性剂诱导的解折叠来检测突变体的稳定性。除了突变体Cys81Ser外，其他突变体与野生型（wt）MIF在构象上有密切相似性，且突变体的稳定性主要归因于酸性pH条件。通过突变体Cys60Ser的近紫外圆二色光谱证实了CALC区域内分子内二硫键的形成。突变体Cys81Ser不参与二硫键形成，但稳定性降低。氧化还原酶分析和MIF特异性细胞因子检测表明，只有活性位点残基的取代会导致MIF失活。突变体Cys60Ser没有酶活性且细胞因子活性显著降低，而突变体Cys81Ser在两项检测中均有活性。Trx样变体显示出显著的酶活性，但活性低于wtMIF；PDI样MIF无酶活性。然而，这两种变体均具有完全的细胞因子活性。连同突变体Cys60Ser较低但非零的细胞因子活性，这表明MIF的细胞因子活性可能不受氧化还原效应的严格调控，或者存在一种可区分的受体机制。本研究为CALC基序在MIF的氧化还原酶和细胞因子活性中的作用提供了证据，并表明Cys81可能介导构象效应。这些突变体的可得性和特性分析应能极大地有助于进一步阐明这种特殊细胞因子MIF的作用机制。
