Molecular Cloning and Characterization of the Mouse Peroxiredoxin V Gene

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Biochemical and Biophysical Research Communications 270, 356–362 (2000)

doi:10.1006/bbrc.2000.2430, available online at http://www.idealibrary.com on

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olecular Cloning and Characterizationf the Mouse Peroxiredoxin V Gene

ae-Hoon Lee,* Sun Jung Kim,* Sang Won Kang,† Kyung-Kwang Lee,*ue Goo Rhee,† and Dae-Yeul Yu*,1

Laboratory of Animal Developmental Biotechnology, Korea Research Institute of Bioscience and Biotechnology,.O. Box 115, Yusong, Taejon 305-600, Korea; and †Laboratory of Cell Signaling, National Heart, Lung,nd Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

eceived March 2, 2000

The peroxiredoxin (Prx) family, thiol-specific antiox-ihptitait

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We have cloned two cDNA isoforms as well asenomic sequences of the mouse Prx V gene andharacterized their molecular genetic features. Twosoforms of the mouse Prx V cDNA were identifiedrom liver and testis. The testis-originated long tran-cripts had extra 1164-bp 5*-UTR sequences com-ared to the liver-originated short transcripts.rimer extension and sequence analyses revealed

hat the two isoforms were presumably transcribedt the same gene locus. The gene was composed of sixxons spanning 3.2 kb. The short transcript wasbundantly expressed in the kidney, liver, and heartf the adult mouse tissues and in the extra-embrane of the 10.5 dpc embryos. The long tran-

cript of 1985 bp was abundantly detected in testisith trace amounts in other tissues. Interestingly, in

estis and fetus, only mRNA expression of the longorm was identified. However, the protein expres-ion was not found in testis, implying that the longorm could not properly direct the protein expres-ion. The long Prx V cDNA has eight uORFs in thextra 5*-UTR, which proceed the major ORF. Thenability of protein expression for the long-formDNA in testis suggests that the uORFs might inhibitranslation of the major ORF and thereby confer theissue-specific regulation of the mouse Prx V gene.2000 Academic Press

Key Words: Peroxiredoxin V; antioxidant enzyme;acterial artificial chromosome; upstream open read-ng frame.

The nucleotide sequence data reported in this paper will appear inenBank, EMBL, and DDBJ Nucleotide Sequence Databases underccession Nos. AF208729 and AF208730.Abbreviations used: Prx, peroxiredoxin; TSA, thiol-specific antiox-

dant; ORF, open reading frame; BSA, bovine serum albumin; dpc,ays postcoitum; uORF, upstream open reading frame.

1 To whom correspondence should be addressed. Fax: 82-42-860-608. E-mail: [email protected].

356006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

dant, can degrade both hydrogen peroxide and alkylydroperoxides (1) and influence the differentiation,roliferation, activity and survival of a range of cellypes (2–4). Since they also exhibit peroxidase activityn a thioredoxin-dependent manner, they are referredo as peroxiredoxin. The biochemical characteristicsnd the evidence from cultured animal cell studiesndicate that the Prx is one of main players in main-aining the cellular redox potential.

Six distinct mammalian peroxiredoxin isozymesave been isolated from various tissues: Prx I (NKEF-A/AG), II (NKEF-B), III (MER5/AOP-1), IV (AOE372),(AOEB166), and 1-Cys Prx (AOP-2). Prx I has heme

inding capacity and has an activity as a natural killerell-enhancing factor (3, 6, 7). The second member ofhe Prx family, Prx II, is able to act as a thiol-specificntioxidant (TSA) and is also able to act as a naturaliller cell-enhancing factor like Prx I (8). Prx III wasriginally reported to be an indispensable factor in-olved in the differentiation of mouse MEL cells (9).he fourth member of family, Prx IV, has been cloned

rom human and rat cells and it is able to act as auppressor of NF-kB activation (10). The Prx V haseen identified recently from human bronchoalveolaravage fluid (11) and describe function. The last one ofrx family, 1-Cys Prx, was also recently cloned viaheir antioxidant activity from human cells (4). Theenomic DNA sequences and its expression were re-orted in cases of Prx I (12), Prx II (13) and I-Cys Prx14, 22) by our lab. and by other research groups. Forhe Prx V gene, human and rat cDNA sequences haveeen reported by Knoops et al. (11). Our colleagueseported the genomic locus of Prx V, PRDX-VI. The Prx

was located in a chromosome 19 with Prx III oferoxiredoxin family (20). In this report, we haveloned two cDNA isoforms as well as the genomic se-uence of the mouse Prx V gene and assayed the ex-ression characteristics of the cDNA isoforms.

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Vol. 270, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ATERIALS AND METHODS

Cloning of cDNA and genomic DNA fragments. A mouse liver andtestis cDNA library in the Uni-ZAP XR vector (Stratagene) were

creened using partial sequence of the human Prx V cDNA as arobe. Positive plaques were isolated, and the cDNA inserts in theBluescript SK(2) plasmids were excised from the Uni-ZAP XRector following the supplier’s instruction. For the cloning of the Prxgene, a BAC library of the 129 SvJ mouse strain was screened by

ustom screening (Genome System Co.). The candidates of Prx Venomic DNA were obtained by hybridization screening with mouseestis Prx V cDNA (821 bp) as a probe. The genomic DNA fragmentsbtained from these clones were digested with EcoRI, BamHI, orhoI. The positive fragment was subcloned into pBluescript SK(2)-ased vectors. These plasmids were sequence analyzed using a seriesf oligonucleotide primers defined initially by the cDNA sequencesnd subsequently by the derived sequences. Restriction enzymeaps were generated by digestion with indicated restriction en-

ymes in Fig. 3 and by Southern blot analysis.

Northern blot and primer extension analyses. For Northern blotnalysis, total RNA was prepared from tissues of a hybrid mousetrain (C57BL/6 3 CBA) according to the standard method (15).orty micrograms of total RNA was separated by size on a 1%garose gel in the presence of 6.7% formaldehyde, transferred to aylon membrane (Roche Molecular Biochemicals), and hybridizedith [32P]-labeled mouse Prx V cDNA probe as described previously

13). Primer extension analysis was carried out as described previ-usly to detect the expression levels and to map the 59 end generatedy RNase processing of the unlabeled substrate RNA (19). The usedrimers (#35 and #36) were underlined in Figs. 2 and 5.

FIG. 1. Alignment of human, rat, and mouse Prx V amino acid seqnd mouse (mPrxV) shows highly conserved amino acid sequences.ndicated.

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Western blot analysis. For the preparation of protein, each organas suspended in homogenization buffer (10 mM Hepes-NaOH (pH.0), 5 mM MgCl2, 15 mM KCl, 1 mM phenylmethylsulfonyl fluoride,eupeptin (5 mg/ml), and aprotinin (5 mg/ml)), and then homogenizedently with a Dounce homogenizer until disruption. The proteinoncentration was monitored using a protein assay kit (Pierce Co.).otal proteins were isolated by centrifugation of the homogenate at2,000g for 1 h. Fifty microgram of each fraction was separated by5% SDS-polyacrylamide gel electrophoresis and transferred to aitrocellulose membrane (Schleicher & Schuell), and subjected to

mmunoblot analysis with a human Prx V antibody. Immune com-lexes were detected with a horseradish peroxidase-conjugated sec-ndary antibody and ECL reagents (Amersham Pharmacia Biotech).

ESULTS

solation of Prx V cDNA Isoforms

A mouse liver and a testis cDNA library in a Lambdani-ZAP XR were screened with the human Prx V

DNA. Two isoforms of Prx V cDNA were isolated, 821p of short form originated from liver and 1985 bp ofong form from testis. An ORF of 627 bp was found bothn the two types of cDNA. Analysis of the deducedmino acid sequences revealed that liver and testisDNA had identical amino acid sequences. The aminocid sequences downstream from the second methio-ine showed high homology among the human, rat,

ces. Clustal W alignment of Prx V from human (hPrxV), rat (rPrxV),ntical residues (asterisks) and conservative substitution (dots) are

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nd mice (77% identity, Fig. 1). However, the aminocid sequences diverged upstream from the second me-hionine. These sequences between Met1 and Met52–53

isplay a feature of the mitochondrial presequence11). The sequences of the murine Prx V gene between

et1 and Met48 also have putative mitochondrial tar-eting sequences and they are composed of amino acidesidues appearing abundantly with positive charges,ew with negative charges, and frequently with hy-roxylation. The arginine residue at position 210 (rel-tive to the NH2-terminal residue of the mature pro-ein) in rat and mice was conserved like many other

FIG. 2. Nucleotide and deduced amino acid sequences of the mound the position and size of the introns are shown. The 11 indicatoly(A)1 signal sequence is underlined. The amino acid sequence forndicated at right.

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itochondrial precursor proteins. The SQL peroxiso-al targeting sequence at the C-terminal was also

onserved among the mammalian species.The 1,985 bp of the testis-originated cDNA has long

1,164 bp) 59-UTR sequence which has several shortpen reading frames. Aside from the 59-UTR se-uences, the remaining downstream region of both theiver and testis form shared identical cDNA sequences.oth sequences contain a poly(A) tract at the 39-end, anpen reading frame of 630 bases, and a 154-base long9-UTR sequence containing the polyadenylation sig-al, AATAAA (Fig. 2).

rx V gene. Exon/intron organization of the Prx V gene is presented,the transcription initiation site of the liver originated mRNA. Theputative ORF is presented in a single letter code with the positions

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loning and Characterization of Mouse Prx VGenomic Fragment

To clone the genomic sequence of the mouse Prx Vene, a mouse BAC library derived from 129/SvJ miceas screened using the 821 bp cDNA as a probe, andne clone (BACM-278b15) that strongly hybridizedith the cDNA probe was finally obtained. Southernlot analysis identified a 6.5kb BamHI fragment,hich was hybridized strongly with the cDNA probe.he DNA fragment was then subcloned into the pBlue-criptII SK(2) vector and subjected to DNA sequencingsing universal primers and oligonucleotides based onhe cDNA sequence. The mouse Prx V gene contains anRF that can be translated into a polypeptide of 209mino acid residues, and shares 100% homology withts cDNA sequences. The results revealed that thelone was an authentic genomic copy for the mouse Prx

gene and it consisted of six exons and five intronsFigs. 2 and 3). All of the splice donor/acceptor siteequences were in accordance with the consensus ‘GT-G’ rule (Table 1). Figure 3 shows a restriction enzymeap of the genomic DNA containing the Prx V gene

FIG. 3. Structure of the murine Prx V gene. A schematic diagramf the Prx V gene structure was superimposed on the restriction-nzyme map. The exons (filled boxes) and the introns (lines) arerawn in scale. The arrow indicates fragment range of clones. Theestriction enzymes used are P, PstI; H, HindIII; E, EcoRI; B,amHI; K, KpnI; X, XbaI; O, XhoI; M, MluI restriction sites.

Exon and Intron Junction Sequences of the mPrxV Gene

ExonNo.

Exonsize(bp)

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Amino acidinterrupted

59 splicedonor

39 splicedonor

1 197 TCAAGgtgac ctcagGTGGG 1,400 —2 134 CTAAGgtgag ctcagACCCA 308 R3 130 GCAAGgtgag tgcagGTTCG 261 —4 39 GGAAGgtgag cctagGCGAC 112 —5 61 AAAAGgtaaa ggcagGTTCT 112 R6 245 CTTTA

Note. Exon and intron sequences are indicated as capital and lowerase, respectively. The 59-donors “gt” and the 39-acceptors “ag” arenderlined. The sites of exon 1 and exon 6 are calculated fromosition 11 and to the end of the liver cDNA, respectively.

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nd its flanking sequence. The entire length of the Prxgene is approximately 3.2 kb (Fig. 3).

xpression Profile of the Prx V Gene

Northern blot analysis, using the liver cDNA as arobe, showed that 821 bp of the short form Prx VRNA was transcribed in all investigated tissues ex-

ept testis with high expression in the kidney, heart,rain, and liver tissues (Fig. 4B). In testis, only theong form of transcripts was expressed with an equalxpression level with the short form in the liver. Thewo variants were also detected in tissues of developingmbryos (Fig. 4A). The short and long forms wereainly expressed in extra-embryonic membranes and

mbryonic bodies, respectively, and their expressionas increased in parallel at 10.5 days of gestation. Toxamine whether the two forms could induce the pro-ein expression, Western blot analysis was carried outor the proteins of adult mouse tissues. The 821 bp

FIG. 4. Expression analysis of the Prx V gene. (A) Northern blotnalysis of embryonic (F) and extra-embryonic (E) tissues of devel-ping embryos. Ten micrograms of total RNA was size-fractionatedn a 1% denaturing agarose gel. The Prx V transcript was detected byybridizing with [32P]-labeled Prx V cDNA fragment cloned from theouse liver library. (B) A mouse multiple tissue Northern blot con-

aining 40 mg of total RNA per each lane was also hybridized with32P]-labeled Prx V cDNA fragment. Different levels of the 821 or223 bp Prx V transcripts are observed in the adult tissues: brain (B),ung (L), spleen (S), kidney (K), liver (Li), testis (T), heart (H), andalivary gland (Sa). (C) Western blot analysis of Prx V proteins.rotein samples (50 mg of the total soluble fraction of various tissues)ere fractionated by SDS-PAGE on a 15% gel, transferred to aitrocellulose membrane, and subjected to immunoblot analysis withhuman Prx V antibody.

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ranscript was translated in proportion to the tran-criptional levels; however, the 1985 bp transcript inestis was not translated at all (Fig. 4C).

To identify the transcription initiation site for theong Prx V mRNA in testis, primer extension analysisas performed. Transcription initiation site of the longrx V was located 21192 bp from the translation ini-

iation site of the major ORF (Figs. 5 and 6B).hereas, transcription of the short Prx V was initiated

t –325 bp from the translation initiation site (Fig. 2).lthough the two cDNAs have different transcription

FIG. 5. The 59-UTR sequence of the testis-originated Prx V tranderline indicates a transcription initiation site of the short cD

nitiation site (open boxed) of major ORF in Prx V and conceptual trresented in a single letter code with the positions indicated at righ

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nitiation sites, their transcripts were presumably en-oded from a single gene because the 59-UTR of theong form was mapped on the just 59 proximal region ofhe short form (Fig. 5). In testis and liver, we comparedhe expression level of mRNA by primer extensionnalysis in addition to Northern blot analysis. Longrx V transcripts were highly expressed in testis al-hough its expression was rarely detected in liver (Fig.A). The short transcripts were highly expressed iniver and it was properly translated to Prx V proteinFig. 4C).

ript. 11 indicates the transcriptional start site of the long cDNA.. The sequences from the transcriptional start site to translationlation of all eight uORFs are shown. The amino acid sequences are

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ISCUSSION

This study was carried out to elucidate the structuref the mouse Prx V cDNA as well as its genomic se-uence, and to characterize the expression profile inouse adult tissues and embryos. We have previously

eported the genomic structure of the mouse Prx I, II,nd 1-Cys Prx (12–14). They have five (1-Cys Prx) or sixPrx I and II) exons, and the mouse Prx V gene identi-ed in this study was composed of six exons. The Prx Vene may exist as a single copy in mouse genomeudged by Southern blot analysis of the chromosomalNA (data not shown). The entire transcription unit of

he Prx V gene is contained in a 6.5 kb BamHI frag-ent. In contrast, the Prx I, II, and 1-Cys Prx consist offamily with several copies of pseudogenes (12–14).

he functional copy of other Prxs has shown similarenomic structures to that of the Prx V gene.The murine Prx V gene was tissue specifically tran-

cribed into two forms of different size, 821 bp and,985 bp mRNA, and they were ubiquitously expressed

FIG. 6. Primer extension analysis of the Prx V mRNA. (A) Com-arison of long and short mRNA expression in testis and liver withhe #36 primer. (B) Transcription initiation sites of the testis-riginated Prx V mRNA. 11 indicates the transcription initiationite of the long transcripts. The sequence of the #35 primer is un-erlined in Fig. 2. Sequencing analysis was carried out with primer36 in Fig. 5.

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ues. Of the two forms of mRNA, only the short tran-cript was translated to protein. It is interesting thatlthough 1985 bp mRNA could not direct any proteinxpression, the mRNA expression level was suddenlyncreased in fetus at 10.5 dpc of developmental em-ryos. The expression of the 821 bp mRNA was alsoncreased in the extra-embryonic tissue at this embry-nic stage. We previously reported Prx I expression inice and in developing embryos (12). The Prx I was

ighly expressed in kidney and liver in adult tissues,nd its expression was suddenly suppressed in embry-nic bodies after 8.5 days’ gestation. The PrxII wasighly expressed in various adult tissues and develop-

ng embryos in a similar level (13). In contrast, highxpression of the Prx V mRNA was detected in theidney, heart, brain and liver tissues and the expres-ion level was variable during the embryo develop-ent. The PrxII was consistently expressed through-

ut the embryo stages (13), whereas the expression of Prxwas suddenly increased at 10.5 dpc embryo stage.In a few mRNAs of the eukaryotic genes, one or moreUGs, or small uORFs that precede the major ORFave been unusually found (21, 23, 24). These AUGs ormall ORFs usually inhibit translation of the down-tream sequences, although some cases have been de-cribed where the uORFs stimulated translation of theajor ORF. When the upstream AUGs of the RARb2

ene were mutated, the expression of the mutant con-tructs in transgenic mice was differently regulated ineart and brain (23). In an in vitro study, cell-freeranslation of the complement factor B (Bf ) mRNA thatas four uORFs in the 59-UTR region revealed that theate of translation of the short form was about two-foldigher than that of long form. Mutation of all uORFsenerated a long transcript with a translational ratequal to that of the short transcript (24). These studiesuggest that the uORFs play a control in the tissue-pecifically and/or developmentally regulated gene ex-ression. The long Prx V cDNA characterized in thistudy is such a transcript with an extra 59-UTR (1164p) that contains eight uORFs preceding the majorRF. Based on the characterization of the testis-riginated long Prx V form both at mRNA and proteinevels, it is speculated that the small uORFs inhibitedhe translation of the major ORF in testis.

Taken together, the results suggest that each mem-er of the Prx family shows its antioxidant activities byeacting with hydrogen peroxide in the embryos anddult tissues through different tissue-specific regula-ion and the Prx V is further regulated at the transla-ional level by several uORFs.

CKNOWLEDGMENT

This work was supported by a grant (HS2490) from the Koreainistry of Science and Technology.

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Molecular Cloning and Characterization of the Mouse Peroxiredoxin V Gene