Heterotrimers formed by tumor necrosis factors of different species or muteins
Heterotrimers formed by tumor necrosis factors of different species or muteins
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DOI：
10.1074/jbc.m104486200
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发表时间：
2001-07-20
影响因子：
4.8
通讯作者：
Brouckaert, P
中科院分区：
文献类型：
--
作者：
Ameloot, P;Declercq, W;Brouckaert, P
以亚纳摩尔浓度孵育鼠肿瘤坏死因子（mTNF）导致三聚体部分解离，同时生物活性降低。使用尺寸排阻色谱法，我们观察到，标记的mTNF的单体的转化不仅是防止通过共孵育与过量的未标记的mTNF，但也与未标记的人TNF（hTNF）。此外，共孵育后的mTNF和hTNF四个不同的TNF复合物，揭示了通过天然聚丙烯酰胺凝胶电泳，即同源三聚体的mTNF和hTNF，以及两个复合物的中间迁移模式。分析凝胶过滤结合天然聚丙烯酰胺凝胶电泳和Western印迹免疫检测表明，这些新的复合物由异源三聚体TNF分子。我们的结论是，交换单体发生在两个不同种类的TNF，这导致同源三聚体和异源三聚体TNF共孵育。为了评估体外受体相互作用，使用mTNF与标记的hTNF（其仅结合mTNF受体I）或标记的突变蛋白mTNF 75（对mTNF受体II特异性）孵育后获得的TNF异源三聚体分子。这些异源三聚体被两种mTNF受体保留，这意味着掺入异源三聚体复合物中的mTNF亚基仍然可以结合两种类型的TNF受体。此外，突变蛋白mTNF 75，这是在L929细胞毒性试验中无活性的存在，表明异源三聚体可以影响整体的生物活性，防止在亚纳摩尔浓度的预孵育过程中的mTNF生物活性的逐渐下降。
Incubation of murine tumor necrosis factor (mTNF) at subnanomolar concentrations results in partial dissociation of the trimers, coinciding with a decrease in bioactivity. Using size-exclusion chromatography, we observed that the conversion of labeled mTNF to monomers is not only prevented by coincubation with an excess of unlabeled mTNF but also with unlabeled human TNF (hTNF). Moreover, after coincubation of mTNF and hTNF four different TNF complexes were revealed by native polyacrylamide gel electrophoresis, viz. homotrimeric mTNF and hTNF, as well as two complexes with an intermediate migration pattern. Analytical gel filtration in combination with native polyacrylamide gel electrophoresis and Western blot immunodetection indicated that these new complexes consisted of heterotrimeric TNF molecules. We conclude that an exchange of monomers takes place during coincubation of two different species of TNF, which results in homotrimeric and heterotrimeric TNF. To assess receptor interaction in vitro, TNF heterotrimeric molecules were used as obtained after incubation of mTNF with labeled hTNF (which only binds to mTNF receptor I) or with labeled mutein mTNF75 (specific for mTNF receptor II). These heterotrimers were retained by both mTNF receptors, which means that the mTNF subunits incorporated in heterotrimeric complexes still can bind to both types of TNF receptor. In addition, the gradual decrease in mTNF bioactivity during preincubation at subnanomolar concentrations was prevented by the presence of mutein mTNF75, which is inactive in an L929 cytotoxicity assay, indicating that heterotrimerization can influence the overall bioactivity.