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【摘要】 目的 为了探索克氏锥虫的谷胱甘肽依赖的过氧化物酶活性。 方法 使用基因克隆与表达鉴定的方法学对克氏锥虫谷胱甘肽依赖的过氧化物酶基因克隆和表达。 结果 一个全新的、18kDa的克氏锥虫单拷贝过氧化物酶基因被完全克隆到E.coli的表达载体pHrcHis上，并测序。蛋白序列分析表明与基因库谷胱甘肽依赖的过氧化物酶有高度的同源性。其表达蛋白被纯化分离，测定其酶活性显示了良好的活性，并能被亚油酸－氢过氧化物饱和。 结论 结果证实克氏锥虫仍然存在过氧化物酶介导的生物代谢。
【关键词】 克氏锥虫 基因克隆 表达 过氧化酶基因
Cloning and expression of a novel peroxidase from Trypanosome cruzi.
YANG Ya-ming, J. Kelly.
(Yunan Provincial Institute for parasitic Disease, Simao 665000, Yunnan, P. R. China; The Pathogen Molecular Biology Unit, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK)
Abstract：Objective To investigate the activity of glutathione-dependent peroxidase in Trypanosome cruzi (T.cruzi). Methods The gene of glutathione-dependent peroxidase in Trypanosome cruzi (T.cruzi) was cloned and expressed and the genome of T.cruzi was anlayzed. Results A single copy gene from T.cruzi tential to encode a novel 18 kDa peroxidase was cloned into E.coli expression vector pHrcHis and sequenced. Analysis of protein sequence indicated that it has high similarity with glutathione peroxidase from GENEBANK database. A recombinant form of the expressed protein in E.coli has been purified, and the activity of peroxidase assayed showing good activity in the presence of glutathione reductase, and it could be saturated by linoleic acid hydroperoxide. Conclusion This results confirm that there is enzyme-mediated peroxide metabolism in T.cruzi.
Key words：Trypanosome cruzi; Cloning of gene; Expression; Peroxidase
Introduction
The American trypanosome, T.Cruzi is the causative agent of Chagas’disease, a major public health problem in South America with up to 20 million people infected, and 20%～40% of those infected develops chronic Chagas’ disease［1］. There is no immediate prospect of vaccine and treatment drugs (nifurtimox and benznidazole) is only effective during acute stage of the disease and is unsatisfactory due to toxicity and limited efficacy.Somuch research effort is directed at the development of new chemotherapeutic agents.
T.cruzi is exposed reactive oxygen spexies(POS)throughout its life cycle. ROSs such as the superoxide anion(O-2.)and hydrogen peroxide(H2O2) cause cellular damage directly by interacting with protein, lipids, nucleic acids etc. or indirectly via the Fe-mediated Fenton and Haber Weis reaction to generated the highly reactive hydroxyl radical(HO). These biological molecules are subject to attack by ROS leading to cell membrane damage, inactivation of essential enaymes, mutagenesis and damage to DNA repair machinery. In most organisms, catalase and glutathione-dependent peroxidase (GPX) enzymes are responsible for metabolizing peroxides. In the latter case, a low molecular weight thiol trypanothione is the most important component of the non-enzymatic system and trypanothione reductase, a parasite-specific enzyme which is central to thiol metabolism and functions by maintaining trypanothione in its reduced form, appears to be essential for cell viability［2］. In tems of the enzymatic antioxidant system, much remains to be determined. Unlike other eukaryotes, catalase and glutathione-dependent peroxidase activities have been reported to be absent. Instead, peroxide metabolism appears to be mediated by trypanotheione-dependent enaymes that are members of the peroxredoxin family. However，this case is being changed. In T.cruzi distinct cytosolic and mitochondrial peroxiredoxins have been identified. The activity of this enzyme has been show to be trypanothione-dependent, but the tryparedoxin/peroxiredoxin interaction has yet to be confirmed［3］ The recent study also demonstrated that T.cruzi expresses a glutathione-dependent peroxidase, and the enzyme can efficiently metabolise hydroperoxides of fatty acids and phospholipids, but not hydrogen peroxide［4］ According to recent advances of studies, Wilkison.S.R et al［4］ proposed two postulated pathways of trypanothione-dependent peroxide metabolism in trypanosomatids:
Scheme 1 proposed scheme for tryanothione-denpendent peroxide metabolism in tryanosomatids. Trypanothione disulphide (TS2) is reduced to dihydrotrypanothione (T(SH)2) by the NADP-dependent flavoprotein trypanothione reductase(TR). Peroxiredoxins (TPx) reduce hydroperoxide (ROOH) to corresponding alcohol (ROH) at the expense of dihydrotrypanothione, but only in the presence of the thioredoxin-like molecular molecule, tyaredoxin (TXN). Dihydrotrypanothione can interact* with oxidized blutathione (GSSG) viaenzymatic and enzymatic mechanisms to generate reduced glutathione (GSH). TcGPXI reduces hydroperoxides at the presence of GSH.
Recently a second phospholipid hydroperoxide glutathione-dependent peroxidase homologue has appeared on the T.Cruzi EST database (accession no.AI0426). it will be interesting to determined the precise substrate-specificity of this enzyme（which we have designated TcGPXII）and to investigate its role within the parasite life-cycle. Of particular importance will be to determine if TcGPXI and TcGPXII have distinct or overlapping functions. Here we demonstrate that a novel peroxidase gene was successfully cloned and sequencing and obtained efficient expression of the gene.
1 Materials and Methods
1.1 Applification of TcGPXII from Trypanosoma cruzi genomic DNA Oligonucleotide primers This primers is designed to correspond to the 5’-and 3’-ands of the TcGPXII gene. In addition, restriction sites have been incorporated the primers(lower case) to facilitate subcloning of the amplified fragment. 5’-primers:
pGGGagatctGGGCAGCAGCACGGTGTTCGOH(Bg1II). 3’-premers:
pCTCaagcttTCATGCACCCCGTTGCGGCCCOH(HingIII).(Stop codon in bold)
1.1.1 Amplification In a total volume of 50μl contains 25.0μl 2x Reaction Mix （x 10 Tag polyerase buffer. 2mM dNTPs, DMSO, Mg2+）, 2.5μl 5’primer (25pmol), 2.5μl 3’-primer(25pmol), 1.0μl template DNA, 18.0μl sterile H2O, 1.0μl Bio taq DNA polymersase. The add 30.0μl sterile mineral oil, short spin again. The amplification conditions are as follows: denaturation 96℃ for 30sec; annealing 50℃ for 30sec; extension 72℃ for 60sec and for 30 cycles on PCR cycler.
1.1.2 Processing of PCR product Using phenol separates the proteins from PCR product. Using sephade G50 kit purifies DNA from the the aqueous phase as instruction of kit.
1.1.3 Restriction digestion and DNA isolation using GenecleanTM Using Bg1III and HindIII digested the PCR fragment, then cut DNA band from agarose gel and place in sterile 1.5μl eppendof tube. Add 1ml of 6M sodium iodide. Incubate at 56℃ for 10 mins. Add the agarose suspension to 10μl glassmilk and mix by gentle inversion every 2 mins for 10 mins at room temperature. Spin 20 seconds in microfuge. Discard supernatant and resuspend pellet in 250μl ice cold new wash solution, and repeated 4 times. Discard supermatant and resuspend pellet in 10μl sterile water. Incubate 56℃ for 5 mins and spin 20 secs. Keep supernatant. Resuspend pellet in 10μl sterile water, and incubate 56℃ for 5 mins, spin again, transfer supernatant to tub containing fist elution. Discard the glassmik pellet.
1.2 Transformation Add 100μl E.Coli (competent cell) into ligation tube (10μl DNA fragment above, 20μl Bglll/HindIII cut expression vector PRC-HisC, 5μl H2O, 2μl 10x ligation buffer, 1μl T4 DNA ligase (5units). Incubate at room temperature for at least 1 hour. Then spread these bacteria onto the agar plate, and then incubate at 37℃ overnight.
1.3 TcTPXII gene sequencing
1.3.1 Plasmid DNA extraction Inoculate 3ml NZCYM broth containing 50μg/ml-1 amplicillin with two colonies from the transformation plate. Incubate the culture overnight at 37℃. using by Alkaline lysis metheod extracted plasmid DNA, the run agarose gel to confirm.
1.3.2 Cycle Sequencin of TcGPX2 gene Using the forward (F) and reverse(R) oligonucleotide primers sets up 2 sequencing reactions (4.0μl “Bigdye”Terminator Ready Reaction mix, 4.4μl DNA template; primer F or R 1.6μl(1.6pmol) and overlay with 40ul of mineral oil). Run thermal cycling as follows for 25 cycle: 96℃ for 10 sec; 50℃ for 5 sec; 60℃ for 3mins. Precipitation of extension products above using ethanol, dry under vacuum, suspend the pellet in 5μl of loading buffer and incubate at 95℃ for 3 minutes, and then cool on ice. The products were sequenced by the ABI 377 automated DNA sequencer.
1.3.3 Analysis of the TcGPX2 DNA sequence After the DNA sequence was edited,usingBLASTN and BLASTX programs compared the similarity with other sequence in the tools of CLUSTALW in www.ebi.ac.uk.
1.4 Restriction digestion of Trypanosoma cruzi genomic DNA Set up the reactions in a total volume of 20μl, using 12μl of T.cruzi (CL Brener) genomic DNA (cloned) and 1μl of EcoRI, SacII and HindIII with 7μl of 1x buffer in a separate tube; short spin. In cubate at 37℃ at least 2 hours. Running on 0.8% agarose gel. Stained by ethidium bromide and observed observed result. Set up standard Southern blot with 32p radiolabelling probes［5］.
1.5 Expression and purification of recombinant TcGPXII Aliquot 3ml NZCYM containing 50μg/ml-1 ampicillin into a uviversal tube. Inoculate the medium with 300μl of the recombinant E.coli culture, and incubate the culture at 37℃ for 2hrs. add 5μl isoproy1 B-D-thiongalactoside(IPTG) (final concentration 0.5mM) and incubate for a further 2～3 hrs at 37℃. Transrer 0.5ml tube and microfuge at top speed for 5min. Resuspend the pelliet in 75μl PBS with a cooktail of protease inhibitors, and lyse the cells by freeze thawing. Repeat twice more. Spin it again, transfer 50μl supernatant to a fresh 1.5ml centrifuge tube for analyzing the GPXII protein by western blotting (using the antibody that is a mouse anti-Xpress-Horseradish peroxidase conjugated antibody diluted 1:5000 as marker).
1.6 Glutathione-dependent peroxidase activity of GPXII Glutathione-dependent peroxidase activity was measured by monitoring NADPH oxidation［4］ Brief description: A standard reaction mixure (ml) containing 100mM Tris/HCL, pH8.0;0.5mM EDTA; 1mM sodium azide; 1.4 units glutathione reductase; 100μM NADPH; 3mM GSH, 50μg GPX2 (purified by a NTA-affinity column)［4］ The reaction was incubated at room temperature for 5 min. The different concentrations of the peroxide, linoleic acid hydroperoxide (0.625～20μM) were assayed in a spectrophotometer in absorbance at 340nm.
2 Results
2.1 Cloning and sequencing of a TcGPX2 gene According to a DNA sequence which is associated with phospholipids hydroperoxide glutathione-denpendent peroxidease homologue from the T.cruzi EST database (accession no.AI0426), DNA primers corresponding to the putative start and stop codons was desigened and amplified the entire open reading frame from T.cruzi genome DNA. About 500bp fragment was obtained (figure 1) and cloned into the vector pTrcHIS-C. This target fragment was sequenced and the inferred amino acid sequences compared with other GPXs family (figure 2).
Analysis of the TcGPX2 protein sequence found four regions characteristic of the GPX family antioxidant enzymes (figre2, regions 1,2,3 and 4), and it is very interested that each region contains the amino acid cysteine residues, particularly, two regions (1 and 2) surrounding the conserved cysteine residue. It has been proven that such residues joined in the catalytic activity of thes anzymes［6］. The T.cruzi enzyme shows the cysteine substitution in region 3 and 4 (figure 2).
Figure 1 PCR product of a T.Cruzi TcGPX2（略）
About 500bp fragment was Were analysed by south indicated. Size was measured by 1 Kb DNA ladder Marker.
Figure 2 Sequence alignment of T.Cruzi GPXII with other protein from this family（略）
LACLC :GLUTATHIONE PEROXIDASE ACCESSION P032770;PLASMODIUM FALCIPARUM : GLUTATHIONE PEROXIDASE gi 1419724;Schistosoma mansoni : glutathine peroxidase gi 2006278A; MOUSE: PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE,MITOCHONDRIAL PRECURSOR(PHGPX),ACCESSION P36970; HUMAN: PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE, MITOCHONDRIAL PRECURSOR(PHGPX),gi 13124748; BOVIN: GLUTATHIONE PEROXIDASE (GSHPX-1),ACCESSION P00435Dashes represent gaps in the sequence made to optimise thealignments. The residues that are similar the TcGPX2 sequence are represented by dots;The star represent identity. Difference between the sequence whencompared with the TcGPX2 are indicated.Boxes represent the regions containing cysteine residuces.
2.2 The genomic organization of TcGPX2 The genomic organization of TcGPX2 was determined by Southern hybridization. The result showed that this was a single-copy gene per haploid genome. For SaII and EcoRI restriction enzymes, two bands were detected (figure3) These indicated there are two allelic forms in the gene corresponding to the two restrictions respectively.
Figure 3 Genomic organisation of TcGPX2（略）
T.Cruzi genomic DNA digested with the enzymes EcoRI(lane 1), SalI (lane 2) and HindIII (lane 3) ern hybridisation usingTcGPX2 as a DNA probe. Size given are in bp.Original agarose gel is showed at right.
2.3 Expression of TcGPX2 gene To investigate the activity of the putative peroxidase. After TcGPX2 was amplified and cloned into the express vector pTrcHis-C.E.coli XL-1 Blue cells were transformed (pTrcHis-GPX2). In this system, the expressed protein is tagged at its N-terminal with a histidine-rich sequence and an epitope detectable with the anti-Xpress monoclonal antibody. After induction with IPTG, an 18 kDa band was expected to detect in the solublefraction of lysate by Western-blot annlysis. Unfortunately, all of us did not detect these expected results, except positive control came out normal result.
2.4 Enzyme activity To determine whether TcGPX2 activity could be saturated by linoleic acid hydroperoxide, assays were carried out using various concentrations of linoleic acid hydroperoxide with a fixed amount of GSH, glutathione reductase and GPX2. The TcGPX2 activities could be rapidly saturated by using linoleic acid hydroperoxide (20μM). GPX2 activity were 1/［linoleic acid hydroperoxide］, which showed a linear relationship (figure 4) From this plot we determined the Km for peroxide to be 0.714M(1/1.4) and Vmax for PEROXIDE TO BE ABOUT 0.0133 umol of NADP oxidized min-1 mg(1/75). When using linoleic acid hydroperoxide. This showed TcGPX2 had biochemical function of the peroxide-metabolising activity.
3 Discussion
The T.cruzi Genome Project was initiated with WHO support［8］. The main work is placed on the sequencing of several thousand expressed sequence tags (ESTs) and provide a large numger of useful information for studying gene transcript and ecpression. Duo to some key enzymatic activities, like catalase and glutathione-dependent peroxidase activities, have not been detected in T.cruzi, theis circumvent biologists to understand how T.cruzi handle the toxic radical which can be produced by immune mechanism or drug metabolism. However, this situation is being changed. The distinct cytosolic and mitochondrial peroxiredxins and glutathione-dependent peroxidase activity has been identified［3］ Comparison to the database, recengly a phospholipids hydroperoxide glutathione-dependent peroxidase homologue has been fund in the T.cruzi EST database (accession no.AI046216) To resolve this paradox, using E.coli expression vector pTrcHis, we successfully cloned the TcGPX2 and had the gene efficiently expressed. The strong experimental evidence below demonstrated this conclusion:(1) around 500bp expected amplification was obtained from T.cruzi genome by specific PCR(figure1). (2) The TcGPX2 protein sequence was analysed by computing compared with the peroxidses in plant, and found that the the Tcgpx2 is high similar to glutathione-dependent resulting multiple sequence alignment (3) TcGPX2 protein was expressed (date did not show enzymatic biochemical pathway involving a glutathione-dependent peroxidase(schme1 upper part) work.
Figure 4 Analysis of TcGPX2 activity with different concentrations of Linoleic acid hydroperoxide.(Lineweaver-Burk, Plot)［7］（略）
TcGPX2 activity was assayed by NADP oxidation in the presence of 50ug GPX2, 3mM reduced glutathione and 1.4 units glutathione reductase. The reaction was initiated by the addition of peroxide (0.625～20uM) for linoleinacid hydroperoxide, TcGPX2 activity is expressed as umol of NADP oxidized min-1 mg of protein-1 while linolein acid hydroperoxide in uM.
When the common molecular characteristics obvious from the sequence alignment in database, GPXs can be more precisely delineated into three major clades:(1)PthecGPXFamily with its side branch giGPX;(2)pGPX together with GPX homologues in which the selenocysteine residue is replaced by cysteine both characterized as secracterised as secreted proteins by typical signal peptides,and (3)th3e PHGPX family［4］.TcGPX2 shows highest similarity to the PHGPX group (figure 2).A cysteine residue has been proven that it plays a important role in the sntioxidant function of the GPX enzymes［4］. Integion 1,fot “NVASK/LCG” sequence expect schistosoma deletion,all these enzymes have cysteine residues,but TcGPX2 have higher change than other one. Comparison with TcGPX1［4］,two regions surrounding the conserved cystein residues are different between “NTASLCS” of TcGPX2 and “NVASRCGYT”OF TcGPX1 (fig2,region 1and2).In addition to TcGPX2 change CN of FPCNQF in TcGPX1 into CA.Interestedly,Plasmodium (CN to TS ) change this structure too.In region 3,TcGPX2 have the most intensive chsnges in sequence.Not only F of “WNF”is substituted by Y, but also turn up a C. In region 4, the LL sequences are replaced by AC. However ,for both TcGPX1(22) and TcGPX2, no distinct structure of 1-cys peroxidoxins has been found［10］ These characteristics are not clear under this limited xperimental data.
TcGPX2 is structurally associated with the glutathione petoxidase family of antioxidant enzymes (figure 2).In this experiment,Km values for peroxide were determined when using a saturated concentration (20uM) of linoleic acid hydroperoxide.The peroxide-metabolising activity of TcGPX2 indicatid that it mediated glutathione-dependent metabolism (figure 4).This result supports the new model (scheme 1) for enzyme-mediated peroxide metabolism system in T. cruzi,like TcGPX1［4］.With trypanosomatids,glutathione reductase activity has not yet been detected and glutathione is maintained in its reduced form by interaction with trypanothione,this can took place in non-enzymatically or via the activity of the a thiol transferase［10～12］ Here glutathione is reduced and make TcGPX2 in its reduced active form (scheme 1).However,this activity is very simple so that it is not enough to show biochernical function of this the localization of the TcGPX2 in T. cruzi to determine whether this enzyme is secreted or housekeeping［3,13］.
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作者单位：云南省寄生虫病防治所，云南 思茅 665000; The Pathogen Molecular Biology Unit，London School of Hygiene & Tropical Medicine，Keppel Street, London WC1E 7HT, UK