Rapid purification and characterization of L-dopachrome-methyl ester tautomerase (macrophage-migration-inhibitory factor) from Trichinella spiralis, Trichuris muris and Brugia pahangi
Rapid purification and characterization of L-dopachrome-methyl ester tautomerase (macrophage-migration-inhibitory factor) from Trichinella spiralis, Trichuris muris and Brugia pahangi
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DOI：
10.1042/bj3350495
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发表时间：
1998-11-01
影响因子：
4.1
通讯作者：
Meyer, DJ
中科院分区：
文献类型：
--
作者：
Pennock, JL;Behnke, JM;Meyer, DJ
巨噬细胞迁移抑制因子（macrophagmigrationinhibition factor， MIF）是哺乳动物t淋巴细胞依赖性适应性免疫的重要刺激物，因此MIF同源物可能作为一种免疫颠覆策略被感染性生物表达。由于MIF活性催化左旋多巴胺甲酯的互变异构化（使用多巴胺互变异构酶），因此通过分析和表征这种活性来研究MIF同源物在几种寄生蠕虫中的发生。在旋毛虫（4期幼虫）和毛线虫（成虫）以及pahangi丝线虫（成虫）的可溶性部分中发现了MIF同源物（dopachrome tamautoerase）的证据。利用苯基琼脂糖层析纯化了tri . muris （TmMIF）和B. pahangi （BpMIF）的MIF同源物，使其具有同质性，螺旋体T. spiralis （TsMIF）的MIF同源物需要进一步的步骤：阳离子交换FPLC。线虫MIF的反相HPLC保留时间和SDS/PAGE M-r值与人MIF相似。TsMIF和TmMIF的n端序列（19个残基）与人类MIF的同源性分别为47%和36%。BpMIF的n端序列（14个残基）与B. malayi （Swiss-Prot, P91850）基因组中的MIF同源物相同，与人类或TsMIF的同源性为43%。TsMIF的多巴胺互变酶活性比其他来源的MIF高10倍。TsMIF、BpMIF和TmMIF的酶活性对血红素（I-50分别为> - 15 μ M、> - 15 μ M和2.6 μ M）的抑制敏感性低于人MIF （I-50 - 0.2 μ M）。在多回线虫和巴西尼波线虫、小膜膜绦虫、曼氏血吸虫、日本血吸虫和血球绦虫，以及含有mif相关基因的自由生活线虫秀丽隐杆线虫的可溶性部分中，未检测到显著的多巴色胺互变酶或苯琼脂糖纯化的mif样蛋白。
Macrophage-migration-inhibition factor (MIF) is an essential stimulator of mammalian T-lymphocyte-dependent adaptive immunity, hence MIF orthologues might be expressed by infectious organisms as an immunosubversive stratagem. Since MIF actively catalyses the tautomerization of the methyl ester of L-dopachrome (using dopachrome tautomerase), the occurrence of MIF orthologues in several parasitic helminths was investigated by assaying and characterizing such activity. Evidence of MIF orthologues (dopachrome tautomerase) was found in the soluble fraction of the nematodes Trichinella spiralis (stage 4 larvae) and Trichuris muris (adults), and the filarial nematode Brugia pahangi (adults). The MIF orthologues of Tr. muris TmMIF) and B. pahangi (BpMIF) were purified to homogeneity using phenyl-agarose chromatography, that of T. spiralis (TsMIF required a further step: cation-exchange FPLC. Retention time on reverse-phase HPLC and M-r on SDS/PAGE of the nematode MIFs were similar to those of human MIF. N-terminal sequences (19 residues) of TsMIF and TmMIF showed 47 and 36% identity, respectively, with human MIF. The N-terminal sequence of BpMIF (14 residues) was identical to that of an MIF orthologue in the genome of B. malayi (Swiss-Prot, P91850) and showed 43 % identity to either human or TsMIF. TsMIF had 10-fold higher dopachrome tautomerase activity than MIF from the other sources. The enzyme activities of TsMIF, BpMIF and TmMIF were less sensitive to inhibition by haematin (I-50 : > 15 mu M, > 15 mu M and 2.6 mu M, respectively) than that of human MIF (I-50 0.2 mu M). Significant dopachrome tautomerase or phenyl-agarose-purifiable MIF-like protein was not detected in the soluble fraction of the nematodes Heligmosomoides polygyrus and Nippostrongylus brasiliensis, the cestode Hymenolepis diminuta, or the trematodes Schistosoma mansoni, S. japonicum and S. haematobium, or the free-living nematode, Caenorhabditis elegans, which does contain an MIF-related gene.