Getting Stephen J, Mahoney David J, Cao Thong, Rugg Marilyn S, Fries Erik, Milner Caroline M, Perretti Mauro, Day Anthony J
Department of Biochemical Pharmacology, The William Harvey Research Institute, St. Bartholomew's and the Royal London School of Medicine and Dentistry, London EC1M 6BQ, United Kingdom.
J Biol Chem. 2002 Dec 27;277(52):51068-76. doi: 10.1074/jbc.M205121200. Epub 2002 Oct 24.
TSG-6 protein (the secreted product of the tumor necrosis factor-stimulated gene-6), a hyaluronan-binding protein comprised mainly of a Link and CUB module arranged in a contiguous fashion, has been shown previously to be a potent inhibitor of neutrophil migration in an in vivo model of acute inflammation (Wisniewski, H. G., Hua, J. C., Poppers, D. M., Naime, D., Vilcek, J., and Cronstein, B. N. (1996) J. Immunol. 156, 1609-1615). It was hypothesized that this activity of TSG-6 was likely to be mediated by its potentiation of inter-alpha-inhibitor anti-plasmin activity (causing a down-regulation of the protease network), which was reliant on these proteins forming a stable, probably covalent approximately 120-kDa complex. Here we have shown that the recombinant Link module from human TSG-6 (Link_TSG6; expressed in Escherichia coli) has an inhibitory effect on neutrophil influx into zymosan A-stimulated murine air pouches, equivalent to that of full-length protein (which we produced in a Drosophila expression system). The active dose of 1 microg of Link_TSG6 per mouse (administered intravenously) also resulted in a significant reduction in the concentrations of various inflammatory mediators (i.e. tumor necrosis factor-alpha, KC, and prostaglandin E(2)) in air pouch exudates. Link_TSG6, although unable to form a stable complex with inter-alpha-inhibitor (under conditions that promote maximum complex formation with the full-length protein), could potentiate its anti-plasmin activity. This demonstrates that formation of an approximately 120-kDa TSG-6.inter-alpha-inhibitor complex is not required for TSG-6 to enhance the serine protease inhibitory activity of inter-alpha-inhibitor. Six single-site Link_TSG6 mutants (with wild-type folds) were compared for their abilities to inhibit neutrophil migration in vivo, bind hyaluronan, and potentiate inter-alpha-inhibitor. These experiments indicate that all of the inhibitory activity of TSG-6 resides within the Link module domain, and that this anti-inflammatory property is not related to either its hyaluronan binding function or its potentiation of the anti-plasmin activity of inter-alpha-inhibitor.
TSG-6蛋白（肿瘤坏死因子刺激基因-6的分泌产物）是一种主要由以连续方式排列的Link和CUB模块组成的透明质酸结合蛋白，先前已证明它在急性炎症的体内模型中是中性粒细胞迁移的有效抑制剂（Wisniewski, H. G., Hua, J. C., Poppers, D. M., Naime, D., Vilcek, J., and Cronstein, B. N. (1996) J. Immunol. 156, 1609 - 1615）。据推测，TSG-6的这种活性可能是由其增强α-抑制剂间抗纤溶酶活性（导致蛋白酶网络下调）介导的，这依赖于这些蛋白质形成稳定的、可能是共价的约120 kDa复合物。在此我们已表明，来自人TSG-6的重组Link模块（Link_TSG6；在大肠杆菌中表达）对酵母聚糖A刺激的小鼠气囊肿中的中性粒细胞流入具有抑制作用，与全长蛋白（我们在果蝇表达系统中产生）的作用相当。每只小鼠静脉注射1 μg Link_TSG6的有效剂量也导致气囊肿渗出液中各种炎症介质（即肿瘤坏死因子-α、KC和前列腺素E2）的浓度显著降低。Link_TSG6虽然无法与α-抑制剂间形成稳定复合物（在促进与全长蛋白形成最大复合物的条件下），但仍能增强其抗纤溶酶活性。这表明，TSG-6增强α-抑制剂间丝氨酸蛋白酶抑制活性并不需要形成约120 kDa的TSG-6·α-抑制剂间复合物。比较了六个单位点Link_TSG6突变体（具有野生型折叠）在体内抑制中性粒细胞迁移、结合透明质酸和增强α-抑制剂间活性的能力。这些实验表明，TSG-6的所有抑制活性都存在于Link模块结构域内，并且这种抗炎特性与其透明质酸结合功能或其增强α-抑制剂间抗纤溶酶活性均无关。
