摘要
活性氧（ROS）是指化学性质活跃的含氧原子或原子团的总称。机体内的ROS是正常生理代谢的产物，不同浓度的ROS可对机体产生有益或有害的影响。低浓度的ROS具有抵御传染性病原体的侵袭、参与细胞信号转导、刺激细胞增殖以及调控细胞凋亡等作用。然而，当各种诱因使机体内ROS产生过多或清除不利时，过量的ROS可以破坏细胞的脂质、蛋白质或DNA并抑制其正常功能，这种现象被称为氧化应激。氧化应激与机体衰老和多种疾病的发生密切相关。机体内有完整的抗氧化体系，包括酶类和非酶类抗氧化剂，其在维持体内ROS产生与清除的平衡中具有重要作用。
硒是人体内不可或缺的微量非金属元素，人体内的硒元素大部分是以Sec的形式掺入到蛋白质中。目前已在人体中发现了25种硒蛋白，这些硒蛋白呈现出不同的组织特异性与亚细胞定位，并在维持人体正常生理功能上发挥着重要作用。其中含硒谷胱甘肽过氧化物酶（GPx）是在抗氧化防御系统中起到重要作用的一类硒蛋白。含硒GPx的活性中心是被称为第21种氨基酸的硒代半胱氨酸（Sec），其与谷氨酰胺（Gln）和色氨酸（Trp）组成的催化三联体结构在GPx家族成员中高度保守。Sec插入到蛋白质中需要一种特殊的机制，尽管真核和原核生物Sec的合成及插入机制并不完全相同，但均需要一类被称为硒代半胱氨酸插入序列（SECIS）的顺式作用元件以及多种反式作用转录因子的协助。也正是因为这种差异性，使得很难利用原核表达系统制备真核含硒蛋白。此外由于天然含硒GPx来源有限且性质不稳定，人们尝试对其进行人工模拟，以期获得具有GPx活力的模拟物代替天然GPx。
为大量制备具有较高活力的GPx模拟酶，我们以hGSTZ1c-1c作为蛋白骨架，通过缺陷型原核表达系统制备多株含硒hGSTZ1c-1c及其突变体蛋白，并结合理论化学的方法对其结构与功能进行研究。而后，利用缺陷型原核表达系统直接制备出重组含硒GPx4蛋白，阐述了非催化中心的Sec替代Cys对蛋白结构产生的影响，并提出减小该影响应采取的策略，成功获得具有较高活力的重组人源GPx4突变体蛋白。
（1）基于定点突变和计算机模拟研究Seleno-hGSTZ1c-1c的结构与功能
天然GPx催化双底物反应，在谷胱甘肽（GSH）存在时能够催化分解多种氢过氧化物，但对底物GSH具有高度特异性。多年来对GPx模拟物的研究也表明，具有GSH结合位点是制备高活力GPx模拟物的一个关键因素。同天然GPx一样，hGSTZ1也是一种催化双底物反应的蛋白酶，且二者拥有共同的底物GSH，因此hGSTZ1可作为一种理想的骨架蛋白模拟GPx。hGSTZ1的催化中心是距离GSH结合位点较近的由Ser14-Ser15-Cys16氨基酸残基组成的SSC基序。前期研究中，通过化学修饰法向已形成固有三维结构的hGSTZ1中引入Sec，获得具有较高活力的含硒模拟酶，且Sec-14和Sec-15被认为是其催化中心。为大量制备高活力的含硒hGSTZ1模拟酶以及研究含硒及非含硒hGSTZ1结构与功能的关系，我们利用缺陷表达法将Sec引入到hGSTZ1拟催化位点中，并利用计算机模拟出hGSTZ1不同突变体的3D结构。实验结果表明Ser-14对GSH的定位具有重要作用，而Ser-15对GST活力的贡献更倾向于它的催化功能，这可能是利用缺陷表达法制备的含硒蛋白Seleno-hGSTZ1c-1c（S14C/S15C）并没有显示出预期的GPx活力的重要原因。此外，多个Cys向Sec的转化会使蛋白质结构发生变化，进而影响与GSH的结合。这可能是Seleno-hGSTZ1c-1c及其突变体蛋白没有显示出较高GPx或GST活力的另一个原因。由此可以看出，催化位点Sec的引入应选择对结构影响较小的氨基酸残基作为候选突变位点；而在使用半胱氨酸缺陷型原核表达系统制备具有GPx活力的含硒模拟酶时，应选择含有较少的Cys的骨架蛋白，防止因多个Sec的引入导致预期之外的结构变化。
（2）重组Se-GPx4的制备及其结构与活性的研究
成熟GPx4是单体蛋白且无糖基化位点，避免了过多的翻译后修饰过程，而缺陷表达系统已被证实能够高效的向蛋白质中引入Sec，因此我们选择利用该系统实现对人GPx4蛋白的异源重组表达。为避免重组GPx4因相互聚集而失活，将Cys-10和Cys-66编码序列突变为Ser编码序列。利用半胱氨酸缺陷型大肠杆菌高效地表达出重组GPx4并对其进行纯化，获得了性质稳定且纯度较高的重组Se-GPx4蛋白。然而重组Se-GPx4蛋白的活力（22U/μmol）虽然比小分子模拟物Ebselen（0.99U/μmol）高，但却比天然GPx4低两个数量级。重组Se-GPx4中除活性中心Sec-46外还有因缺陷表达系统对蛋白质中Cys的非选择性替换而插入的5个非催化Sec。其所在位置无法通过GSH保持其还原状态，因此这部分Sec是以亚硒酸态存在于蛋白质中。通过计算机模拟出重组Se-GPx4的3D结构，将其与模拟天然GPx4蛋白结构进行比较并分析。由于Sec的结构与Cys差别较大，这种替换引起了蛋白质结构的变化，进而导致重组Se-GPx4催化中心Sec-46中羟基的指向与天然GPx4不同，可能会在空间上妨碍与GSH的结合。在整个催化反应中，次硒酸与GSH的快速反应在防止GPx的催化基团因过度氧化而失活中起到至关重要的作用，GSH对次硒酸还原效率的降低可能是重组Se-GPx4蛋白活力降低的直接原因。本研究首次利用原核表达系统制备人源重组Se-GPx4蛋白酶，虽然活力并没有达到天然酶水平，但在开发GPx药用价值中迈出了坚实的一步，对GPx异源表达的研究亦有重要意义。
（3）重组GPx4突变体的改造与活力改善
除活性中心以外的多个Sec的引入会引起蛋白结构的变化，进而影响其活力。因此提高重组Se-GPx4活力最有效的方法是对其进行改造，使其空间结构可能地接近天然GPx4。与Cys理化性质最接近的氨基酸是Ser，因此使用Ser将重组GPx4中的Cys替换不会对其蛋白构象产生较大影响。本实验利用快速定点突变法依次将GPx4蛋白中的Cys-148、Cys-107、Cys-37、Cys-75和Cys-2突变为Ser，并通过半胱氨酸缺陷型大肠杆菌向各个突变体中引入Sec。实验结果表明，随着GPx4蛋白中非催化中心Sec数量的减少，Se-GPx突变体的活力具有增大的趋势。当重组Se-GPx4中除活性中心Sec-46以外的所有Sec被Ser取代后，该突变体呈现出最高的GPx活力值（646U/μmol），大约是突变前GPX活力的15倍。在对重组Se-GPx4完全突变体的3D结构进行分析后发现，其“催化三联体”结构与天然GPx4相近，且次硒酸态Sec的活性羟基指向了蛋白外侧。该结构可能是只保留活性中心Sec-46的Se-GPx4突变体活力提高的原因。此外，本实验将半胱氨酸缺陷型大肠杆菌表达系统与SPP低温表达系统进行联合，并使用“联合”表达方法向重组GPx4蛋白中引入Sec。与单独缺陷表达相比，“联合”表达方法被证明具有更高的可溶性含硒蛋白产率以及更低的目的蛋白表达成本。本实验进一步证实多个Sec的引入可导致GPx结构变化，进而对酶活力产生负面影响。同时也印证了使用原核表达系统制备真核GPx4蛋白时，可以通过Ser代替Cys以减小因非特异性引入的多个Sec与Cys结构差异而导致的蛋白整体结构变化所产生的影响，继而提高其酶活力。这对今后GPx蛋白的重组表达具有重要意义，同时也为GPx结构与催化机制的研究奠定了基础。
Free radicals can be defined as molecules or molecular fragments containing oneor more unpaired electrons in atomic or molecular orbitals. Reactive oxygen species(ROS) are intermediate products of normal metabolism and they can be either harmfulor beneficial to living systems. ROS at low/moderate concentrations playphysiological roles in defending against infectious agents and regulating signaltransduction pathways and transcription factors. The harmful effects of ROS occur athigh concentrations and the excess ROS can cause damage to lipids, proteins andnucleic acids in cells, causing the condition termed oxidative stress. Oxidative stresshas been implicated in a number of human diseases as well as in the ageing process.The balance between beneficial and harmful effects of ROS is essential for thesurvival of organisms and achieved by the antioxidant action of non-enzymatic andenzymatic antioxidants.
Selenium is well established as an essential trace mineral of fundamentalimportance to human health and this element is mainly incorporated into thepolypeptide chain as part of selenocysteine (Sec). At least25proteins are identified sofar which have different tissue specificity and maintain normal physiological functionof many body tissues. Glutathione peroxidase (GPx) is a member of selenoenzymesand acts as as a free radical scavenger using GSH as reducing substrate. The catalyticcenter of GPx is known as catalytic triad consisting of a Sec, a Trp, and a Gln residue.Sec is also known as the21stamino acid and encoded by UGA, which usuallyfunctions as a stop codon. The incorporation of Sec into protein requires a specialmechanism. The selenocysteine insertion sequence (SECIS) and multi trans-actingfactors are necessary to achieve the incorporation. And the fact that the mechanismdiffers from prokaryotes to eukaryotes makes it difficult to directly heterologouslyexpress recombinant mammalian selenoproteins in E. coli. Due to the disadvantages of limited availability, poor stability and high molecular weight of native GPx, itstherapeutic usage is limited. Researchers’ study focused on artificial imitation of GPx.
In this research, selenocysteine-containing hGSTZ1c-1c and its various mutantswere prepared by using cysteine auxotrophic strain to obtain GPx mimics with highactivity. And the role of different residues in the SSC motif on binding GSH and theeffect of introducing Sec into hGSTZ1c-1c were explored by site-directedmutagenesis and computational analysis. Furthermore, we produced recombinanthuman GPx4mutants using cysteine auxotrophic strain and discussed the impact ofnon-catalytic Sec on the structure of the protein. A strategy was proposed forenhancing the catalytic activity of recombinant enzyme and a new GPx4mutant withhigh activity was obtained.
(1) Characterization of catalytic activity and structure of selenocysteine-containinghGSTZ1c-1c based on site-directed mutagenesis and computational analysis
Glutathione peroxidase (GPx, EC1.11.1.9) is an important class of antioxidantenzymes that protects cells from oxidative damage. As radical scavengers, GPxscatalyze the GSH-dependent reaction and thereby reduce different types of peroxidesto their respective alcohols. Because of the natural binding site of GSH, humanglutathione transferase zeta1c-1c (hGSTZ1c-1c) has been considered to be an idealprotein scaffold for imitating glutathione peroxidase. The SSC motif(Ser14-Ser15-Cys16) highly conserved in most of GSTs was regarded as the activecenter in catalysis. It has been reported that the Sec-14and Sec-15ofselenium-containing hGSTZ1-1(Seleno-hGSTZ1-1) produced by chemicallyconverting Ser to Sec residue contributes significantly to the GPx activity. But it wasdifficult to prepare selenoenzyme in large amount due to the restrictions of chemicalmethod. Instead, Cys auxotrophic expression system was used to produce GPxmimics. The aim of this study is to investigate the changes of catalytic activity fromhGSTZ1c-1c to Seleno-hGSTZ1c-1c produced by using Cys auxotrophic expressionsystem. Furthermore, the role of different residues in the SSC motif on binding GSHand the effect of introducing Sec into hGSTZ1c-1c were explored by site-directed mutagenesis and computational analysis. In this report, several residues near GSHwere mutated to selenocysteine (Sec) or cysteine (Cys) residues and the impacts of thesubstitutions on different activities were discussed. Mutations of Ser-14or/and Ser-15to Cys or Sec residues resulted in dramatic loss of catalytic activity of hGSTZ1c-1cwith chlorofluoroacetic acid (CFA) as substrate, indicating the importance of thehydroxyl groups in Ser-14and Ser-15. And subsequent study by molecular modelingsuggested that Ser-15was probably involved in catalysis, while Ser-14may play acrucial role in binding and orientation of GSH and possibly had a synergistic effectwith Ser-15in catalysis. On the contrary, the result of converting Cys-16to Serindicated its trivial role in catalysis. The investigations of theselenocysteine-containing hGSTZ1c-1c (Seleno-hGSTZ1c-1c) and the mutant S17Cimplied that the substitutions of multi-Sec for Cys residues could lead to subtlechange in the structure of the protein molecule and concomitant change in catalyticactivity as a direct result. This finding provides evidence that the protein scaffoldcontaining fewer cysteines should be chosen for imitating GPx using Cys auxotrophicexpression system to avoid unexpected structural changes.
(2) Prokaryotic expression of recombinant Se-GPx4and study on its structure andactivity
Cytosolic GPx4is a single polypeptide chain protein with a molecular mass of19.5kDa, which does not undergo major post-translational modification. cysteineauxotrophic strain has been proved to efficiently achieve the introduction of Sec intoproteins. In this study, recombinant GPx4was produced by using this E. coli strain.Cys-10and Cys-16were mutated to Ser residues for they may cause the recombinantprotein aggregation. Although recombinant GPx4was obtained with high purity andstability and showed higher GPx activity (22U/μmol) than Ebselen (0.99U/μmol),but two orders of magnitude lower than native GPx4. There were five non-catalyticSec residues besides Sec-46in recombinant GPx4which were unspecificallyintroduced by cysteine auxotrophic strain. And the five Sec were seleninic acidsbecause they could not be reduced by GSH. The substitution of multi-Sec for Cys in recombinant GPx4may lead to structural change for the structural differencesbetween Sec and Cys. And this difference caused the active hydroxyl group of Sec-46orientating toward the inside of the protein in comparison to the putative native GPx4and thereby directly interfered with binding of GSH to the recombinant enzyme. Inthe catalytic reaction, the rapid reactions of the selenenic acid with GSH and of theresulting selenenyl sulfide (E-SeSG) with a second GSH to produce the selenol appearto be very important, because these reactions ensure that the selenium moiety in theenzyme is not irreversibly inactivated. Lower reduction efficiency might be the mainreason for the loss of the activity of recombinant GPx4. In this study, it is the firsttime to prepare recombinant human GPx4in prokaryotic expression system. Althoughthe activity of recombinant GPx4is not as high as native GPx4, but the resultsrepresent a solid step forward on the path toward heterologous expression ofrecombinant GPx in E. coli.
(3) A modification of recombinant GPx4mutant
The substitution of multi-Sec for Cys in recombinant GPx4may lead to structuralchange and thereby cause the loss of the normal GPx activity. So we need an effectiveway to improve the activity by modifying recombinant GPx4. Ser and Cys exhibitvery similar physical and chemical properties and the substitution of Ser for Cys inrecombinant GPx4wouldn’t lead to severe structural change. In this study, Cys-148,Cys-107, Cys-37, Cys-75and Cys-2in GPx4were mutated to Ser in turn and theresponding Se-GPx4mutants were prepared by using the “combined” expressionsystem. The results indicated that the catalytic activity of recombinant GPx4wasenhanced following the number of non-catalytic Sec decreased. The mutant showedthe highest catalytic activity (646U/μmol) when all the Sec residues in recombinantSe-GPx4mutant were replaced by Ser, which was15fold higer than non-mutatedrecombinant GPx4. A three-dimensional structure of this recombinant Se-GPx4mutant was constructed and the catalytic triad was therefore detected. Unlikenon-mutant recombinant GPx4, the active hydroxyl group of E(mutant)-SeOH isorientated toward the same direction compared with the putative native GPx4. And the increase of the catalytic activity of recombinant Se-GPx4mutant may attribute tothis. In addition, a new way to prepare recombinant Se-GPx4was established by usingcysteine auxotrophic strain combined with SPP system. The “combined” expressionsystem has been proved to express selenoprotein with higher efficiency of solubleprotein production and lower cost of protein production than cysteine auxotrophicexpression system. These results indicated the feasibility of substituting Ser for Cys toenhance the catalytic activity of recombinant Se-GPx4produced in prokaryotic host.This research is of great significance for the heterologous expression of recombinantSe-GPx, and lay the foundation for investigating the clear structure and catalyticmechanism of GPx.
引文
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