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具有GSH结合位点的硒蛋白在真核细胞中的表达
中文摘要

硒是人体必需的微量元素之一,它主要以硒蛋白的形式存在于生物体内,并以硒酶的形式发挥其生物学功能,其中最重要的一种含硒酶是谷胱甘肽过氧化物酶(GPX; EC1.11.1.9)。GPX是一种重要的抗氧化剂,在机体抗氧化、清除自由基、防止细胞免受氧化损伤中扮演着重要的角色。然而,由于该酶来源有限、稳定性差,分子量大等因素的制约,大大限制了它的开发和利用。其在临床治疗和预防肿瘤、心脑血管疾病、炎症等疾病方面展示的美好前景,激发人们采取多种方法人工模拟GPX用于其催化机制和药理学研究。 硒代半胱氨酸(Sec)是GPX的催化关键氨基酸,也是在各种抗氧化酶模拟物中引入GPX活力的有效基团。有趣的是,编码它的密码子是通常作为终止子的UGA,硒蛋白的广泛分布表明Sec的正常掺入需要一个特殊的调控机制。在Escherichia coli中, Sec的表达效率只有正常氨基酸的1-3%,因此硒蛋白的体内表达量非常低。而且,Sec插入依赖的原核生物硒代半胱氨酸插入序列(Selenocysteine Insertion Sequence,SECIS)需要紧邻编码Sec的UGA插入到其编码序列中,这势必影响硒蛋白的构象,从而使其丧失活性。然而在真核生物中,SECIS则位于mRNA 3'端非翻译区。 在本研究工作中,我们首先利用硒蛋白真核生物合成元件,探讨异源硒蛋白在体外培养的哺乳动物细胞株中表达的可行性,并利用绿色荧光蛋白报告基因研究了硒蛋白真核表达的通读效率。然后,分别选用了与GPX具有共同催化底物的人Zeta族谷胱甘肽硫转移酶(hGST Z1-1)以及抗GSH的单链抗体2D8作为蛋白骨架,利用真核生物Sec插入机制在酶的活性中心定点引入GPX的催化氨基酸,并通过在硒蛋白真核表达载体中引入前导肽,实现了含硒蛋白在真核细胞中的分泌表达。 本研究首次报道了利用真核生物基因工程方法将具有GSH结合位点的亲本蛋白转换成相应硒蛋白,而且利用这种方法得到的硒蛋白结构明确、催化位点单一。这一成果为进一步制备催化位点明确的小分子高活性的人源GPX模拟物奠定了良好的理论和实验基础。

英文摘要

Selenium is an essential micronutrient for mammals, incorporation into selenoproteins by Selenocysteine (Sec) and displays its physiology function in forms of seleno-cysteine (Sec) containing enzyme. Both of the deficiency and excessiveness of selenium will lead to the incidence of various diseases. Selenocysteine (Sec) is the catalytically active residue of many selenoenzymes which are involved in redox reactions. Sec isp encoded by an opal codon UGA, usually as a stop codon, and a special element is involved in its incorporation into protein. This element is a stem-loop structure, known as the Sec insertion sequence (SECIS). In prokaryotes, the SECIS element is located immediately downstream of the UGA within the open reading frame (ORF) and thereby the SECIS element must be affect the conformation of the expressed protein furthermore lost its activity. Therefore it is difficult to express Sec by traditional recombinant DNA technology in prokaryotes. In eukaryotes, however, the SECIS element is instead located in the 3' untranslated region (3'-UTR) of the selenoprotein-coding mRNA, which does not affect the conformation of the expressed protein. Furthermore, the bacterial SECIS is species-specific, while the SECIS elements in eukaryotes are somewhat less species-restricted. Glutathione peroxidase (GPX, EC 1.11.1.9) is a crucial antioxidant seleno-cysteine (Sec) containing enzyme which functions as an antioxidant to protect biomembranes and other cellular components from oxidative damage by catalyzing the reduction of a variety of hydroperoxides, using glutathione as the reducing substrate. It plays a significant role in protecting cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Since the classical 'cytosolic GPX' was discovered in 1957. seven types of selenium-containing GPXs have been found in vertebrates. Structural determinations, detailed kinetic studies, and modeling of enzyme-substrate complexes have suggested how the selenocysteine residue plays its critical role in the catalytic cycle by utilizing the specific redox properties of selenium. However, due to the limited availability, poor stability and high molecular weight of native GPX, its therapeutic usage is limited. Therefore, artificial imitation of GPX becomes the focus of scientists for its development and application. Several methods have been used to generate GPX mimics for mechanistic study and pharmacological development. However, only few of these methods involved genetic engineering and none of them have achieved specific site-directed incorporation of sec without other modifications. These have hampered further structure-function studies. Evolution of a probable 'glutathione-binding ancestor' resulting in a common thioredoxin-fold for glutathione S-transferases and glutathione peroxidases may possibly suggest that a glutathione S-transferase could be engineered into a selenium-containing glutathione S-transferase (seleno-GST) by incorporation Sec into its activity site, having GPX activity. Based on this presumption, we hope to achieve expression of seleno-GST to imitate GPX using the selenoprotein synthesis machinery of eukaryotes. In this study, we do the following research: 1.GFP as a reporter detecting the read-through of selenocysteine in eukaryote. Since GFP was described as a reporter gene in 1994 for the first time, it has been used extensively as a fusion tag. a reporter, as well as in protein interaction experiments and in fluorescent energy resonance transfer assays.GFP is able to autocatalytically fluoresce without external agents other than oxygen. We investigated the feasibility of GFP as a reporter to detect the read-through of selenocysteine in cells. We developed the p-seGST-green fluorescent protein (GFP) assay vetor as follows: to clone enhanced green fluorescent protein (EGFP) coding sequence into the immediately downstream of the UGA of Segst fragment using overlap PCR, and the fusion gene Segst-gfp was then subcloned to the sites Hind Ⅲ and Sal Ⅰ of pSelExpress1 to construct p-seGST-EGFP. Fluorescence microscopy images of the expression of seleno-GST-green fluorescent protein (GFP) chimaera indicated that we successfully achieved the read-through of a UGA codon to specifically incorporate Sec as residue 16 in hGSTZ1-1. 2.Expression of selenocystein-containing GST in HEK293FT cells. Expression of Sec-containing proteins in mammalian cells, especially at high levels, is challenging. Based on the vector for overexpression of selenoproteins in mammalian cells, we introduced the murine Ig κ-chain leader sequence to develop a vector for expression and and secretion of a Sec-containing protein, designated as pSelE-L, which has Toxoplasma SelT SECIS element, as well as the SECIS-binding protein 2 (SBP2). To test this vector for selenoprotein heterologous expression, we cloned the human GST ORF containing an N-terminal His-tag into pSelE-L and incorporate Sec as residue 16 useing hGST Z1-1 as the parent protein which has the GSH binding site. HEK 293FT cells were transfected with these constructs and were cultured in the media supplementing with sodium selenite. Western blotting showed that the expression of seleno-GST was dose-dependent on selenium. Therefore, we achieved expression and secretion of a Sec-containing protein in HEK293FT cells by supplementing the media with 10μM sodium selenite. The molecular weight of the secreted protein was about 24.2kDa which was consistent with that of the wild type hGSTZl-1. 3.Expression of selenocystein-containing scFv-2D8 in HEK293FT cells. Preparation of selenocystein-containing scFv by chemical modification has some disadvantages such as higher cost, lower yield. Moreover, it is incapable of specifically mutating Ser into Sec on the target location, and in some cases, other hydroxyl groups in the protein are inevitably converted into selenols, which will hamper the further structure studies.For solving these problems, we used the expression system of selenium-containing protein in eukaryotes mentioned above, cloned selenium-containing single chain Fv fragment-2D8 (scFv-2D8) ORF with GSH binding site into pSelE-L, and we achieved expression and secretion of a Sec-containing scFv-2D8 protein in HEK293FT at the same condition which mentioned above. Although we enhanced the amount of selenoprotein expression using this definite expression vector, it is difficult to achieve the same level as the other proteins, and the amount of producted selenoprotein is not enough for purification to detecte the activity of GPX. In conclusion, we have shown that recombinant selenoproteins with incorporation of selenocysteine residues may be heterologously produced by intrducing SECIS element in the 3' untranslated region (3'-UTR) of the target protein in eukaryotes. We thereby believe this system affords several advantages for achieving truly site-directed substitution and further structure-function characterization of the Selenoproteins. Here, we report for the first time the conversion of the parent protein with GSH binding site into seleno-protein by means of genetic engineering in eukaryotes, and the recombinant selenoproteins is single catalytically active residue and well-characterized structure produced by this method. This result will lay a foundation for preparing much smaller GPX mimics with higher activity. Key words: Selenocysteine, GFP, GST, single chain Fv fragment, selenoprotein

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