Bernhagen J, Mitchell R A, Calandra T, Voelter W, Cerami A, Bucala R
Picower Institute for Medical Research, Manhasset, New York 11030.
Biochemistry. 1994 Nov 29;33(47):14144-55. doi: 10.1021/bi00251a025.
The cytokine macrophage migration inhibitory factor (MIF) has been identified to be secreted by the pituitary gland and the monocyte/macrophage and to play an important role in endotoxic shock. Despite the recent molecular cloning of a human T-cell MIF, characterization of the biochemical and biological properties of this protein has remained incomplete because substantial quantities of purified, recombinant, or native MIF have not been available. We describe the cloning of mouse MIF from anterior pituitary cells (AtT-20) and the purification of native MIF from mouse liver by sequential ion exchange and reverse-phase chromatography. For comparison purposes, human MIF was cloned from the Jurkat T-cell line and also characterized. Mouse and human MIF were highly homologous (90% identity over 115 amino acids). Recombinant mouse and human MIF were expressed in Escherichia coli and purified in milligram quantities by a simple two-step procedure. The molecular weight of native mouse MIF (12.5 kDa monomer) was identical with that of recombinant mouse MIF as assessed by gel electrophoresis and mass spectroscopy. No significant post-translational modifications were detected despite the presence of two potential N-linked glycosylation sites. Recombinant MIF inhibited monocyte migration in a dose-dependent fashion, and both recombinant and native MIF-exhibited comparable biological activities. MIF induced the secretion of tumor necrosis factor-alpha and stimulated nitric oxide production by macrophages primed with interferon-gamma. Circular dichroism spectroscopy revealed that bioactive mouse and human MIF exhibit a highly ordered, three-dimensional structure with a significant percentage of beta-sheet and alpha-helix conformation. Guanidine hydrochloride-induced unfolding experiments demonstrated that MIF is of low to moderate thermodynamic stability. These studies establish the biochemical identity of native and recombinant MIF and provide a first insight into the three-dimensional structural properties of this critical inflammatory mediator.
细胞因子巨噬细胞移动抑制因子（MIF）已被证实由垂体以及单核细胞/巨噬细胞分泌，并在内毒素休克中发挥重要作用。尽管近期已对人T细胞MIF进行了分子克隆，但由于无法获得大量纯化的、重组的或天然的MIF，该蛋白的生化及生物学特性的表征仍不完整。我们描述了从小鼠垂体前叶细胞（AtT-20）克隆小鼠MIF以及通过连续离子交换和反相色谱法从鼠肝中纯化天然MIF的过程。为作比较，我们还从Jurkat T细胞系克隆了人MIF并对其进行了表征。小鼠和人MIF高度同源（在115个氨基酸上有90%的同一性）。重组小鼠和人MIF在大肠杆菌中表达，并通过一个简单的两步程序纯化出毫克量的蛋白。通过凝胶电泳和质谱评估，天然小鼠MIF（12.5 kDa单体）的分子量与重组小鼠MIF相同。尽管存在两个潜在的N-连接糖基化位点，但未检测到明显的翻译后修饰。重组MIF以剂量依赖方式抑制单核细胞迁移，重组MIF和天然MIF均表现出相当的生物学活性。MIF诱导肿瘤坏死因子-α的分泌，并刺激经γ干扰素预处理的巨噬细胞产生一氧化氮。圆二色光谱显示，具有生物活性的小鼠和人MIF呈现高度有序的三维结构，其中β折叠和α螺旋构象占相当比例。盐酸胍诱导的去折叠实验表明，MIF具有低至中等的热力学稳定性。这些研究确定了天然和重组MIF的生化特性，并首次深入了解了这种关键炎症介质的三维结构特性。
