Domain unfolding plays a role in superfibronectin formation
Domain unfolding plays a role in superfibronectin formation
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DOI：
10.1074/jbc.m509082200
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发表时间：
2005-11-25
影响因子：
4.8
通讯作者：
Erickson, HP
中科院分区：
文献类型：
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作者：
Ohashi, T;Erickson, HP
超纤维连接蛋白(SFN)是一种纤维连接蛋白(FN)聚合体，由FN与FN的III型结构域片段Anastellin混合而成。然而，这种聚集的机制尚不清楚。在本研究中，我们发现Anastellin与Fn以接近4：1的比例共沉淀，Anastellin：Fn单体。Anastellin的主要结合部位在片段(III)1-3，它结合了Anastellin的三个分子，并能够在没有FN分子其余部分的情况下形成沉淀。Anastellin与(III)3的结合引起了该结构域的构象变化，暴露了一个隐秘的热裂解蛋白敏感部位。一个额外的金雀花蛋白与(III)11结合，在那里它促进了(III)11的热分解。(III)3中的一个工程二硫键抑制了聚集和蛋白酶消化，表明(III)3的稳定性是SFN形成的关键因素。我们提出了一个形成SFN的三步模型：1)FN-III结构域自发展开和重新折叠；2)Anastellin结合到一个未折叠的结构域，防止其重新折叠，并使其具有暴露的疏水表面和β-折叠边缘；以及3)这些暴露的元素与其他分子上类似的暴露的元素结合，导致聚集。该模型与我们观察到的凝聚动力学为一级反应，反应时间为500-700 S是一致的，相似的机理可能有助于天然FN基质的组装。
Superfibronectin (sFN) is a fibronectin (FN) aggregate that is formed by mixing FN with anastellin, a fragment of the first type III domain of FN. However, the mechanism of this aggregation has not been clear. In this study, we found that anastellin co-precipitated with FN in a ratio of similar to 4:1, anastellin: FN monomer. The primary binding site for anastellin was in the segment (III)1-3, which bound three molecules of anastellin and was able to form a precipitate without the rest of the FN molecule. Anastellin binding to (III)3 caused a conformational change in that domain that exposed a cryptic thermolysin-sensitive site. An additional anastellin binds to (III)11, where it enhances thermolysin digestion of (III)11. An engineered disulfide bond in (III)3 inhibited both aggregation and protease digestion, suggesting that the stability of (III)3 is a key factor in sFN formation. We propose a three-step model for sFN formation: 1) FN-III domains spontaneously unfold and refold; 2) anastellin binds to an unfolded domain, preventing its refolding and leaving it with exposed hydrophobic surfaces and beta-sheet edges; and 3) these exposed elements bind to similar exposed elements on other molecules, leading to aggregation. The model is consistent with our observation that the kinetics of aggregation are first order, with a reaction time of 500-700 s. Similar mechanisms may contribute to the assembly of the native FN matrix.