A novel plant protein disulfide isomerase family homologous to animal P5 – molecular cloning and characterization as a functional protein for folding of soybean seed‐storage proteins
A novel plant protein disulfide isomerase family homologous to animal P5 – molecular cloning and characterization as a functional protein for folding of soybean seed‐storage proteins
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DOI：
10.1111/j.1742-4658.2007.06199.x
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发表时间：
2008-02
期刊：
影响因子：
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通讯作者：
Hiroyuki Wadahama;S. Kamauchi;Yumi Nakamoto;K. Nishizawa;M. Ishimoto;T. Kawada;R. Urade
中科院分区：
文献类型：
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作者：
Hiroyuki Wadahama;S. Kamauchi;Yumi Nakamoto;K. Nishizawa;M. Ishimoto;T. Kawada;R. Urade
蛋白质二硫键异构酶在新生多肽的折叠和内质网二硫键的形成中起重要作用。在本研究中，我们克隆了一个新的大豆叶片蛋白二硫键异构酶家族基因。CV Jack)mRNA.该基因编码一种名为GmPDIM的蛋白质。它由438个氨基酸组成，其序列和结构域结构与动物P5相似。在大肠杆菌中表达的重组GmPDIM对变性核糖核酸酶A具有氧化复性活性。与拟南芥和水稻的序列比较表明，大豆的外显子/内含子结构具有明显的保守性。GmPDIM基因启动子中的共同序列包含一个顺式作用的调控元件，用于未折叠的蛋白质反应，以及种子特异表达所需的其他调控基序。我们观察到GmPDIM在ER胁迫条件下表达上调，并且在大豆的子叶等组织中普遍表达。它定位于急诊室的管腔。来自免疫共沉淀实验的数据表明，GmPDIM以非共价方式与种子储存蛋白球蛋白的前体丙球蛋白结合。此外，GmPDIM还与α‘亚单位β-伴大豆球蛋白有关，这是一种在衣霉素存在下的种子贮藏蛋白。这些结果表明，GmPDIM可能在贮藏蛋白的折叠中发挥作用，不仅作为一种硫醇氧化脱氢酶，而且还作为分子伴侣发挥作用。
The protein disulfide isomerase is known to play important roles in the folding of nascent polypeptides and in the formation of disulfide bonds in the endoplasmic reticulum (ER). In this study, we cloned a gene of a novel protein disulfide isomerase family from soybean leaf (Glycine max L. Merrill. cv Jack) mRNA. The cDNA encodes a protein called GmPDIM. It is composed of 438 amino acids, and its sequence and domain structure are similar to that of animal P5. Recombinant GmPDIM expressed in Escherichia coli displayed an oxidative refolding activity on denatured RNase A. The genomic sequence of GmPDIM was also cloned and sequenced. Comparison of the soybean sequence with sequences from Arabidopsis thaliana and Oryza sativa showed significant conservation of the exon/intron structure. Consensus sequences within the promoters of the GmPDIM genes contained a cis‐acting regulatory element for the unfolded protein response, and other regulatory motifs required for seed‐specific expression. We observed that expression of GmPDIM was upregulated under ER‐stress conditions, and was expressed ubiquitously in soybean tissues such as the cotyledon. It localized to the lumen of the ER. Data from co‐immunoprecipitation experiments suggested that GmPDIM associated non‐covalently with proglycinin, a precursor of the seed‐storage protein glycinin. In addition, GmPDIM associated with the α′ subunit of β‐conglycinin, a seed‐storage protein in the presence of tunicamycin. These results suggest that GmPDIM may play a role in the folding of storage proteins and functions not only as a thiol‐oxidoredactase, but also as molecular chaperone.