Investigation of the mechanism of iron release from the C-lobe of human serum transferrin: Mutational analysis of the role of a pH sensitive triad
Investigation of the mechanism of iron release from the C-lobe of human serum transferrin: Mutational analysis of the role of a pH sensitive triad
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DOI：
10.1021/bi027071q
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发表时间：
2003-04-08
期刊：
影响因子：
2.9
通讯作者：
Mason, AB
中科院分区：
文献类型：
--
作者：
Halbrooks, PJ;He, QY;Mason, AB
转铁蛋白（transferrin，TF）是广泛分布于脊椎动物中的一个蛋白质家族，在脊椎动物中，它们在铁结合和转运中起主要作用。大多数TF由两个同源的叶组成，N叶和C叶，每个叶能够结合单个铁原子。人血清转铁蛋白（human serum transferrin，hTF）与血液中的铁结合并将其递送至活跃分裂的细胞;通过受体介导的内吞作用过程，血清中的二铁hTF（pH值接近7.4）与细胞表面的特异性TF受体结合并被内化，于是内体中的pH值下降（pH值接近5.6）促进铁的释放。许多因素影响铁的释放速率，包括pH、螯合剂、温度、盐和叶-叶相互作用。我们和其他人已经积极研究了从hTF的重组N-叶释放铁的机制;相反，从C-叶释放铁的确切细节仍然没有得到很好的表征，但似乎与N-叶中发现的那些不同。最近，为了简化纯化方案，我们表达并纯化了含有N-末端六组氨酸标签的全长重组hTF [Mason等（2002）Biochemistry 41，9448-9454]。在目前的工作中，我们表达了一个全长重组hTF含有K206 E突变，使N-叶不容易释放铁。由此产生的全长hTF使我们能够专注于C端，并研究引入C端的突变的影响。这种策略的成功记录和体外诱变用于确定三个残基的C-叶是铁释放的关键。虽然这一三联体的重要性已被明确证实，但还需要进一步的研究来完全阐明铁从hTF的C叶释放的机制。此外，在本工作中进一步记录了增加盐浓度对hTF的两个叶的铁释放的影响的显著差异。
The transferrins (TFs) are a family of proteins that are widely distributed in vertebrates, where they serve a major role in iron binding and transport. Most TFs are composed of two homologous lobes, the N- and C-lobes, each able to bind a single iron atom. Human serum transferrin (hTF) binds iron in the blood and delivers it to actively dividing cells; through the process of receptor-mediated endocytosis, diferric hTF in the serum (pH similar to7.4) binds to specific TF receptors on the cell surface and is internalized, whereupon a pH drop in the endosome (pH similar to5.6) facilitates iron release. Many factors affect the rate of iron release, including pH, chelator, temperature, salt, and lobe-lobe interactions. We, and others, have actively studied the mechanism of iron release from the recombinant N-lobe of hTF; in contrast, the exact details of iron release from the C-lobe have remained less well characterized but appear to differ from those found for the N-lobe. Recently, to simplify the purification protocol, we have expressed and purified full-length recombinant hTF containing an N-terminal hexahistidine tag [Mason et al. (2002) Biochemistry 41, 9448-9454]. In the present work, we have expressed a full-length recombinant hTF containing a K206E mutation such that the N-lobe does not readily release iron. The resulting full-length hTF allows us to focus on the C-lobe and to study the effects of mutations introduced into the C-lobe. The success of this strategy is documented and in vitro mutagenesis is used to identify three residues in the C-lobe that are critical for iron-release. Although the importance of this triad is unequivocally demonstrated, further studies are needed to completely elucidate the mechanism of iron release from the C-lobe of hTF. In addition, the striking difference in the effect of increasing salt concentrations on iron release from the two lobes of hTF is further documented in the present work.