Evidence that clusterin has discrete chaperone and ligand binding sites
Evidence that clusterin has discrete chaperone and ligand binding sites
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DOI：
10.1021/bi0157666
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发表时间：
2002-01-08
期刊：
影响因子：
2.9
通讯作者：
Wilson, MR
中科院分区：
文献类型：
--
作者：
Lakins, JN;Poon, S;Wilson, MR
白藜芦醇是第一个被鉴定的哺乳动物细胞外伴侣蛋白，并与多种部分未折叠的应激蛋白结合。胆固醇还结合许多不同的非应激配体，包括细胞表面受体低密度脂蛋白受体相关蛋白-2（LRP-2）。目前还不清楚丛生蛋白是否通过一个或多个结合位点与所有这些配体结合。此外，这些结合相互作用中涉及的丛生蛋白区域仍有待鉴定。作为这些问题的调查的一部分，我们表达重组人clusterin在酵母毕赤酵母。所得蛋白质具有α链的C-末端区域和β链的N-末端区域的可变蛋白水解截短。我们比较了分子伴侣和配体结合活性的重组产品与那些从人血清中纯化的clusterin。我们还测试了clusterin与配体的结合是否可以通过与其他clusterin配体的竞争性结合或通过抗clusterin单克隆抗体来抑制。总的来说，我们的结果表明：（i）丛生蛋白具有三个独立的类的结合位点的LRP-2，应激蛋白，和未应激的配体，分别和油的LRP-2和应激蛋白的结合位点可能是在分子的部分以外的α-链的C-末端区域或β-链的N-末端区域。已经表明，在体内，丛生蛋白结合细胞外环境中的毒性分子，并将这些分子携带到表达LRP-2的细胞中进行摄取和降解。这一假设得到了我们的支持，表明clusterin具有离散的LRP-2和其他（潜在的毒性）分子的结合位点。
Clusterin is the first identified extracellular mammalian chaperone and binds to a wide variety of partly unfolded, stressed proteins. Clusterin also binds to many different unstressed ligands including the cell surface receptor low-density lipoprotein receptor-related protein-2 (LRP-2). It is unknown whether clusterin binds to all of these many ligands via one or more binding sites. Furthermore, the region(s) of clusterin involved in these many binding interactions remain(s) to be identified. As part of an investigation of these issues, we expressed recombinant human clusterin in the yeast Pichia pastoris. The resultant protein had variable proteolytic truncations of the C-terminal region of the alpha-chain and the N-terminal region of the beta-chain. We compared the chaperone and ligand binding activities of this recombinant product with those of clusterin purified from human serum. We also tested whether the binding of clusterin to ligands could be inhibited by competitive binding with other clusterin ligands or by anti-Clusterin monoclonal antibodies. Collectively, our results indicate that (i) clusterin has three independent classes of binding sites for LRP-2, stressed proteins, and unstressed ligands, respectively, and Oil the binding sites for LRP-2 and stressed proteins are likely to be in parts of the molecule other than the C-terminal region of the alpha-chain or the N-terminal region of the beta-chain. It has been suggested that, in vivo, clusterin binds to toxic molecules in the extracellular environment and carries these to cells expressing LRP-2 for uptake and degradation. This hypothesis is supported by our demonstration that clusterin has discrete binding sites for LRP-2 and other (potentially toxic) molecules.