Molecular cloning,expression, and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage

A cDNA (C2C-Prx) corresponding to a 2Cys-peroxiredoxin (2Cys-Prx) was isolated from a leaf cDNA library of Chinese cabbage. The predicted amino acid sequence of C2C-Prx has 2 conserved cysteines and several peptide domains present in most of the 2Cys-Prx subfamily members. It shows the highest sequence homology to the 2Cys-Prx enzymes of spinach (88%) and Arabidopsis (86%). Southern analysis using the cDNA insert of C2C-Prx revealed that it consists of a small multigene family in Chinese cabbage genome. RNA blot analysis showed that the gene was predominantly expressed in the leaf tissue of Chinese cabbage seedlings, but the mRNA was generally expressed in most tissues of mature plant, except roots. The expression of C2C-Prx was slightly induced by treatment with H₂O₂ (100M) or Fe³⁺/O₂/DTT oxidation system, but not by ABA (50 M) or GA₃ (10 M). The C2C-Prx is encoded as a preprotein of 273 amino acids containing a putative chloroplast-targeting signal of 65 amino acids at its N-terminus. The N-terminally truncated recombinant protein (C2C-Prx) migrates as a dimer in a non-reducing SDS-polyacrylamide gel and as a monomer in a reducing condition. The C2C-Prx shows no immuno cross-reactivity to antiserum of the yeast thiol-specific antioxidant protein, and vice versa. The C2C-Prx prevents the inactivation of glutamine synthetase and the DNA cleavage in the metal-catalyzed oxidation system. In the yeast thioredoxin system containing thioredoxin reductase, thioredoxin, and NADPH, the C2C-Prx exhibits peroxidase activity on H₂O₂.     

Role for plant peroxiredoxin in cadmium chelation

Abstract

Studies on heavy metal underline the role of thiols in plants and attribute tolerance to metal binding. The thiol and peroxiredoxins (Prx) contents and guaiacol peroxidase (GPOX) activity were analyzed in the cotyledons and embryo of pea (Pisum sativum L.) germinating seeds exposed to toxic Cd concentration. The 2-cysteine peroxiredoxin (2Cys-Prx) level as well as the non-protein thiol (–SHNP) pool increased in both tissues treated with Cd compared to the control. An oxidized dimer of 2Cys-Prx was resolved in the presence of Cd ions. The obtained results suggest that Prx constitute a main key target in Cd toxicity. Despite of the decrease in GPOX activity due to the generation of an intracellular oxidative stress, a protective action via increasing Prx expression on thiols is possible to improve the redox status.     

Cloning and characterization of a 2-Cys peroxiredoxin from Pisum sativum

A cDNA sequence coding for a pea (Pisum sativum L.) 2-Cys peroxiredoxin (2-Cys Prx) has been cloned. The deduced amino acid sequence showed a high sequence homology to the 2-Cys Prx enzymes of Phaseolus vulgaris (86%), Arabidopsis thaliana (75%), and Spinacia oleracea (75%), and contained a chloroplast target sequence at its N-terminus. The mature enzyme, without the transit peptide, has a molecular mass of 22 kDa as well as two cysteine residues (Cys-53 and Cys-175) which are well conserved among proteins of this group. The protein was expressed in a heterologous system using the expression vector pET3d, and was purified to homogeneity by three sequential chromatographic steps. The enzyme exhibits peroxidase activity on hydrogen peroxide (H(2)O(2)) and t-butyl hydroperoxide (TBHP) with DTT as reducing agent. Although both pea Trxs f and m reduce oxidized 2-Cys Prx, Trx m is more efficient. The precise conditions for oligomerization of 2-Cys Prx through extensive gel filtration studies are also reported. The transition dimer-decamer produced in vitro between pH 7.5 and 8.0 and the influence of DTT suggest that a great change in the enzyme quaternary structure of 2-Cys Prx may take place in the chloroplast during the dark-light transition. In addition, the cyclophilin-dependent reduction of chloroplast 2-Cys Prx is shown.     

Isolation and Characterization of a New Peroxiredoxin from Poplar Sieve Tubes That Uses Either Glutaredoxin or Thioredoxin as a Proton Donor

A sequence coding for a peroxiredoxin (Prx) was isolated from a xylem/phloem cDNA library from Populus trichocarpa and subsequently inserted into an expression plasmid yielding the construction pET-Prx. The recombinant protein was produced in Escherichia coli cells and purified to homogeneity with a high yield. The poplar Prx is composed of 162 residues, a property that makes it the shortest plant Prx sequence isolated so far. It was shown that the protein is monomeric and possesses two conserved cysteines (Cys). The Prx degrades hydrogen peroxide and alkyl hydroperoxides in the presence of an exogenous proton donor that can be either thioredoxin or glutaredoxin (Grx). Based on this finding, we propose that the poplar protein represents a new type of Prx that differs from the so-called 2-Cys and 1-Cys Prx, a suggestion supported by the existence of natural fusion sequences constituted of a Prx motif coupled to a Grx motif. The protein was shown to be highly expressed in sieve tubes where thioredoxin h and Grx are also major proteins.     

中国明对虾过氧化物还原酶基因在大肠杆菌中的重组表达、产物纯化及活性测定

过氧化物还原酶（Prx）是生物体内广泛存在的一类酶,在消除过氧化氢和抗氧化胁迫中起着重要的作用。本研究采用PCR扩增编码中国明对虾Prx成熟肽的基因,并克隆到大肠杆菌表达载体pCR®T7/NT TOPO® TA中进行体外重组表达。重组质粒转化大肠杆菌BL21 (DE3) pLysS后,经IPTG诱导表达产生包涵体形式的目的蛋白。对重组蛋白进行LC–ESI–MS分析,结果表明融合蛋白的四个肽段与中国明对虾Prx相应肽段完全一致。将重组蛋白通过金属螯合柱进行纯化,进而透析、复性,最后获得了具有较高过氧化物酶活性的重组Prx。中国明对虾Prx的成功表达,为深入研究其在中国明对虾免疫反应和抗氧化胁迫中的作用奠定了基础。     

Biochemical characterization of a novel 2-Cys peroxiredoxin from <Emphasis Type="Italic">Antrodia camphorata</Emphasis>

Peroxiredoxins (Prxs) play important roles in antioxidation and cell signaling. A gene encoding a novel 2-Cys Prx was identified based on sequence homology in an expressed sequence tag database of the Antrodia camphorata, a medicinal mushroom found only in Taiwan. The 2-Cys Prx cDNA (940 bp) encodes a protein of 188 amino acid residues with calculated molecular mass of 20,965 Da and a pI of 5.89. The coding region was subcloned into pAVD10, transformed into Escherichia coli, and expressed as a His-tagged fusion protein. The purified enzyme was characterized under various conditions. The Prx retained 68% activity after being heated at 60°C for 2 min. It was stable under a broad pH range from 5 to 11. The enzyme activity was slightly decreased in the presence of 1% sodium dodecyl sulfate. The enzyme was somewhat susceptible to chymotrypsin treatment but resistant to digestion by trypsin. Jenq-Kuen Huang, Chuian-Fu Ken, and Hui-Ming Huang contributed equally to this paper.     

Purification and cloning of pierisin-2, an apoptosis-inducing protein from the cabbage butterfly, Pieris brassicae.

The cabbage butterfly Pieris rapae contains a strong apoptosis-inducing substance, pierisin, against human cancer cell lines, which is thought to act via ADP-ribosylation. Here we report the purification and cloning of an apoptosis-inducing substance, designated as pierisin-2, from another cabbage butterfly, Pieris brassicae. Pierisin-2 was purified from pupae by sequential chromatography and its cytotoxic and apoptosis-inducing activities to various cancer cells were similar to those of pierisin, designated as pierisin-1, from P. rapae. cDNA cloning of pierisin-2 was performed on the basis of the partial amino-acid sequence. The nucleotide sequence indicated that the cDNA encodes an 850-amino-acid protein with a calculated molecular mass of 97 986. The deduced amino-acid sequence of pierisin-2 was 91% identical with that of pierisin-1. In vitro expressed protein in the reticulocyte lysate exhibited apoptosis-inducing activities against human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, similar to the purified native pierisin-2 from the pupae. Pierisin-2 shows regional sequence similarities with certain ADP-ribosylating toxins such as the A-subunit of cholera toxin. The results from site-directed mutagenesis at Glu165, a conserved residue among ADP-ribosylating enzymes necessary for NAD binding, and from experiments with ADP-ribosylating enzyme inhibitors suggested that pierisin-2 could be considered as an ADP-ribosylating toxin like pierisin-1.     

Expression profiles of peroxiredoxin proteins of the rodent malaria parasite Plasmodium yoelii

Patterns of expression of the 2-Cys and 1-Cys peroxiredoxin (Prx) proteins of the rodent malaria parasite Plasmodium yoelii during its life cycle were observed by immunofluorescent antibody staining and confocal laser scanning microscopy. 2-Cys Prx was expressed in the parasite cytoplasm throughout the life cycle, and the thioredoxin (Trx)-peroxidase activity of 2-Cys Prx revealed with the recombinant protein suggested that the Prx is constitutively expressed and, thus, likely plays a housekeeping role in the parasite's intracellular redox control. In contrast, 1-Cys Prx showed stage-specific expression in blood-stage parasites. The limited expression of 1-Cys Prx in the trophozoite cytoplasm suggests that 1-Cys Prx may be involved in haemoglobin metabolism by the parasite, which generates a prooxidative haem iron and increases intracellular oxidative stress. The antioxidant activity of 1-Cys Prx was tested for its ability to protect yeast enolase against inactivation of the mixed-function oxidation system. Differential expression of the two Prx proteins during the erythrocytic and insect stages suggests the importance of these proteins in protecting parasites against oxidative stress, which is generated by the parasite's metabolism and also from the environment.     

Molecular and functional characterization of a cyclophilin with antifungal activity from Chinese cabbage

An antifungal protein that inhibits the growth of filamentous fungal pathogens was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The N-terminal amino acid sequence of the protein was highly homologous to that of plant cyclophilins and consequently the protein was denoted as C-CyP. To understand the antifungal activity of C-CyP, we isolated a cDNA encoding its gene from a Chinese cabbage leaf cDNA library. The Chinese cabbage genome bears more than one C-CyP gene copy and C-CyP mRNA is highly expressed in all tissues except the seeds. Recombinant C-CyP catalyzed the cis-trans inter-conversion of the Ala-Pro bond of the substrate, which indicates this protein has peptidyl-prolyl cis-trans isomerase activity. It also inhibited the growth of several fungal pathogens.     

Miiuy croaker (Miichthys miiuy) Peroxiredoxin2: Molecular characterization,genomic structure and immune response against bacterial infection

Peroxiredoxin2 (Prx2) protein is an important member in cellular antioxidant protein superfamily. Prx2 exists widely in prokaryotes and eukaryotes, it not only plays a part in eliminate reactive oxygen, but also takes effect in many other metabolic activities, such as stimulate epithelial cell proliferation, participate in the signal transduction in cells and so on. After molecular cloning we got that the complete cDNA sequence of Prx2 consists 882 bp, including a 5′-UTR of 46 bp, an open reading frame (ORF) of 591 bp, and a 3′-UTR of 245 bp. The complete gene of miiuy croaker Prx2 has 5 exons and 4 introns. The deduced 197 amino acid residues of miiuy croaker Prx2 consists a Val-Cys-Pro (VCP) motifs. In order to better elucidate the immune mechanisms of the Prx2 in the lower vertebrates, we conducted a research about the Prx2 gene of miiuy croaker and its expression pattern after bacterial infection. Real-time PCR (RT-PCR) results showed that expression of Prx2 was up-regulated in kidney, liver and spleen under infection with Vibrio anguillarum, and expressed level differently in ten different uninjected tissues. Our results suggested that Prx2 might be involved in immune defence in miiuy croaker.     

Functional analysis and expression characteristics of chloroplastic Prx IIE

Peroxiredoxins (Prxs) are ubiquitous thiol-dependent peroxidases capable of eliminating a variety of peroxides through reactive catalytic cysteines, which are regenerated by reducing systems. Based on amino acid sequences and their mode of catalysis, five groups of thiol peroxidases have been distinguished in plants, and type II Prx is one of them with representatives in many sub-cellular compartments. The mature form of poplar chloroplastic Prx IIE was expressed as a recombinant protein in Escherichia coli . The protein is able to reduce H₂O₂ and tert-butyl hydroperoxide and is regenerated by both glutaredoxin (Grx) and thioredoxin (Trx) systems. Nevertheless, compared with Trxs, Grxs, and more especially chloroplastic Grx S12, are far more efficient reductants towards Prx IIE. The expression of Prx IIE at both the mRNA and protein levels as a function of organ type and abiotic stress conditions was investigated. Western blot analysis revealed that Prx IIE gene is constitutively expressed in Arabidopsis thaliana , mostly in young and mature leaves and in flowers. Under photo-oxidative treatment and water deficit, almost no change was observed in the abundance of Prx IIE in A.   thaliana , while the level of Prx Q (one of the two other chloroplastic Prxs with 2-Cys Prx) increased in response to both stresses, indicating that plastidic members of the Prx family exhibit specific patterns of expression under stress.     

青花菜雄性不育相关基因BoDHAR的克隆与表达分析

以一个与甘蓝显性核不育相关的差异表达片段的序列为信息探针,通过在NCBI与TAIR网站数据库中进行同源EST序列搜索,经人工拼接、RT-PCR、PCR克隆与序列分析,获得了青花菜脱氢抗坏血酸还原酶DHARdehydroascorbatereductase基因的cDNA与DNA全长序列,命名为BoDHAR。并利用双链接头介导PCR的染色体步行技术(genomewalking)克隆了其上游644bp的5′端序列。所获的BoDHAR基因全长1486bp,存在两个内含子,DNA编码区序列633bp,编码210个氨基酸;序列分析表明BoDHAR与同源基因AT1G19570.1cDNA序列有82.3%的一致性,推导的氨基酸序列有79.6%的一致性;编码的水溶性蛋白存在多个磷酸化位点;5′端上游区存在明显的转录调控序列。半定量RT-PCR结果表明BoDHAR在可育系花蕾中的表达量明显高于不育系花蕾,在花药中的表达明显高于其它部位。