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Short Communication Cloning and Sequencing of Mouse Glutaredoxin (grx) cDNA ANTONIO MIRANPA-VIZUETE, JOSE RAFAEL PEDRA JAS, ANASTASIOS E. DAMDIMOPOULOS and GIANNIS SPYROU

Dept. of Biosciences at NOVUM, Center for Biotechnolqyy, Karolinska lnstitutrt, S-34157 Huddinge, Suieden

(Received 13 lanunry, 1999)

Glutaredoxins are small proteins (12 kDa) with a conserved active sequence Cys-Pro-Tyr(-Phe)-Cys that catalyse GSH-disulfide oxidoreduction reac- tions in the presence of NADPH and glutathione reductase. Many mammalian glutaredoxins have been characterized and human and pig cDNA sequence determined. However, no mouse glutare- doxin cDNA or protein sequence has yet been reported. We have cloned a cDNA from a mouse liver library that encodes the putative mouse glutar- edoxin homologue. The deduced polypeptide sequence encodes a 107 amino acid protein display- ing a high degree of homology with other members of the glutaredoxin family.

Database Accession No: AF109314

Glutaredoxins (Grx) and thioredoxins (Trx) comprise a superfamily of low molecular weight proteins that share a common fold (the thiore- doxin fold) and participate as general protein disulfide reductases in a variety of redox proc- esses (Holmgren, 1989). Thioredoxins use NADPH and the flavoenzyme thioredoxin reductase to reduce disulfides in many proteins whereas glutaredoxins are coupled to NADPH,

glutathione reductase and GSH for the same purpose (Padilla et aZ.,1995). Glutaredoxins differ from thioredoxins mainly in the sequence of their redox active site (CPY(F}C for Grx and WCGPC for Trx) althought, in some cases both proteins perform the same reaction, e.g. reduc- tion of PAPS reductase (Tsang M.L.S., 1981) or ribonucleotide reductase (Holmgren, 1976). Glu- taredoxins and thioredoxins are ubiquitous pro- teins and many forms of them exist within the same organism. For example, two thioredoxins and three characterized glutaredoxins (plus another ORF containing the typical active site CPYC) exist in E. coli (Laurent et al., 1964; Miranda-Vizuete et al., 1997; Holmgren, 1979; Wslund et al., 1994); Saccharomyces cerevisiae con- tains three thioredoxins (two cystosolic and one mitochondrial) (Hall et al., 1971; Pedrajas et al., 1998) and nine ORF with the glutaredoxin CPYC active site (Saccharomyces cerevisiae genome data- base); higher organisms like plants or mammali- ans contain three or two thioredoxins, respectively (Besse and Buchanan, 1997; Spyrou et al., 1997) but only one form of glutaredoxin

* # To whom correspondence should be addressed: Giannis Spyrou, Dept. of Biosciences at NOVUM, Center for Biotechnol- ogy, Karolinska Institutet, 5-14157 Huddinge, Sweden. Ph: +46-8-6089162; Fax: +46-8-7745538; email: giannis.spyrou@cbt.ki.se

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TTACCGGACCATTACTTTCAACTGCACGTTCCTCCCTGGAGAAGCTGCAGCCTGTCAGC

A T G G C T C A G G A G T T T G T G A A C TGC A A G A T C C A G T C T GGG A A G G T G G T C G T G T T C A T C A A G M A O E F V N C K I O S G K V V V F I K

CCC A C C T G C C C C T A C T G C A G A A A G A C C C A A G A A A T C C T C A G T C A A C T G C C T T T C A A A C A A P T K T 0 E i L S 0 L P F K 0

G G T C T T C T G G A G T T T G T G G A C A T C A C A G C C A C T A A C A A C ACC A G T G C G A T T C A A G A T T A T G L L E F V D I T A T N N T S A I O D Y

T T A C A A C A G C T C A C C GGA G C G A G A A C A G T T C C T CGG G T C T T C A T A G G T A A A G A C T G C A T A L O O L T G A R T V P R V F I G K D C I

GGC G G A T G C A G T G A T C T A A T C T C C A T G C A A C A G A C T GGG GAG C T G A T G A C T C G G C T G A A G G G C S D L I S M O O T G E L M T R L K

C A G A T T G G A G C T C T G C A G T T A T A A AAGGGGTGGCAGGCAGAGTCCATGCTGACACAGCTGTCTAACCATGC 0 I G A L 0 L a - *

T G A T G G C C A G T G C C C C T G A G A G T T G A T G T G C A T C G C A G A G G A T G T C A G T A T T T C C T G G T G A C T G G G A T T T T T C A A C A A G G C G G C C T T T A T T C T T C T T T T C C T C A G T G C T A A A A A C T G T T G C A A T T T G C C C C T A A C C A T G G G G C C G A G A A G C T T A A C A G A C C A C A C T G G T T T G A T T A T C C A T T C T T C A T G T G C C A A C A T G T C T C T A C C T C T A A G C C C A G G T T T T C C A A A T C C A G T T G C T C T A A A T C T C C A G T G G A T C T G T T G C T G G T T T T C T G C T A C T G T T C G T C A G C T G A A G T C A T T T T G C A G A A G T C C A C T T T C T A A A G A A T T A T T G A A T C A A T G G A T A T C G A A A A T T T G T T T C C T G A G T C A T G C A T C G G C T C C T C T C T C C C T C G T G C A C G C A C C C T T C C C A C T C C T G C A T T C A C T G C C C T T A C T T A G C C A G T G T T C T C A G C C T C A A C C T C C T A C A A C C C G C A G A C G T C C A C A C T G G T G T G A G G A C G C T G T T T G A A A A A T C A G A T G A A C T T T A G C A T A G C T G G T C C T C A C A G A G G C C A C G T T A A C T T A G G C G G C A G A G C A G A T G G T G C A T G C A G C T C C C T C T G T A A A G G T G A T T A A T T G T C C A G A A A A T C C C A A G C A G C T G T G T G T T G A T C C G A G T T A G A G G G C C A G A A A A A T C A A A T G T G A A A C A C A A A A T T G C A A A A T T C T C C T T C C A A G A A T T T C T G T G A A A G A C G T TGTTTCTGAAACATTGTCCTAAACAGTTTCTTCCATCCAAACTTTGACATTTTGCTTTGATGTCTTGCTATGCTGTTTA A T T T T C A T G G A T C T G T A G A T C A C T T C T C T G G T C T C C A G T G A G G A G G A T T C A T T A C T A T T A A A G A T G T A T C T A T A G A T A A A A A A A A A A A A A

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FIGURE 1 cDNA and deduced amino acid sequence of mouse glutaredoxin. Nucleotides are numbered on the left and amino acids on the right. The active site is boxed and the presumptive polyadenylation signal is double underlined. Asterisks indicate the stop codon

(Padilla ef al., 1995). Mammalian glutaredoxin has been purified from many different sources and the amino acid sequence of the human, rab- bit, bovine and pig forms are available (reviewed in Padilla et al., 1995). We report here the cDNA and the deduced amino acid sequence of the mouse form.

The GenBank sequence data base at the National Center of Biotechnology Information was accessed via Internet and the Basic Local Alignment Search Tool (BLAST) was used to identify mouse EST clone-encoded proteins homologous to the human Grx sequence (acces- sion number: X76648) (Padilla et al., 1995). Many EST hits were identified and, being guided by homology with human Grx, a contig sequence (a hypothetical cDNA) was obtained by merging

the overlapping ESTs. To confirm the sequence and actually clone the mouse full-lenght cDNA we designed two specific primers at the flanking regions of the contig and PCR was performed on a mouse liver cDNA library. The resulting PCR product was cloned into the pGEM-Teasy vector (Promega) and three independent clones were sequenced in both strands. The complete sequence of the cDNA (1310 bp) consists of an open reading frame of 320 bp, a 59 bp of 5’-UTR and a 930 bp fragment of 3’-UTR including the poly(Af) (Figure 1). The mouse cDNA sequence is significantly larger than the two so far cloned glutaredoxin cDNAs from human (837 bp) and pig (575 bp) (Padilla et al., 1995; Yang et al., 1989). The open reading frame extends from the methionine at position 60 to the termination

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MOUSE GLUTAREDOXIN cDNA

1 M A Q E F V N C K I Q S G K V V V F I K P T C P Y C R K T Q E I L S Q L P F K Q 1 M A Q E F V N C K I Q P G K V V V F I K P T C P Y C R R A Q E I L S Q L P I K Q

1 M A Q A F V N S K I Q P G K V V V F I K P T C P F C R K T Q E L L S Q L P F K E 1 M A Q E F V N S K I Q P G K V V V F I K P T C P Y C R K T Q E I L S Q L P F K Q

1 M A Q A F V N S K I Q P G K V V V F I K P T C P Y C R K T Q E L L S Q L P F K Q

1x1

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piggrx rabbitgrx

bovinegrx

* * * * * * * * * * * * * * * * * * * I * * * I * * * * * * * * * *

50 60 70 80

41 G L L E F V D I T A T N N T S A I Q D Y L Q Q L T G A R T V P R V F I G K D C I mxLe - g r x 41 G L L E F V D I T A T N H T N E I Q D Y L Q Q L T G A R T V P R V F I G K D C I m g r x 41 G L L E F V D I T A A G N I S E I Q D Y L Q Q L T G A R T V P R V F I G Q E C I bwinegrx 41 G L L E F V D I T A T S D T N E I Q D Y L Q Q L T G A R T V P R V F I G K E C I piggrx 41 G L L E F V D I T A T S D M S E I Q D Y L Q Q L T G A R T V P R V F L G K D C I rabbitgm

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

93 100 hcmslogy B

-grx sl G G C S D L I S M Q Q T G E L M T R L K Q I G A L Q L 81 G G C S D L V S L Q Q S G E L L T R L K Q I G A L Q 89.6 - g r x 81 G G C T D L V N M H E R G E L L T R L K Q M G A L Q 83.0 bwine gm

81 G G C T D L E S M H K R G E L L T R L Q Q I G A L K 82.1 Pi9 grx 81 G G C S D L I A M Q E K G E L L A R L K E M G A L R Q 85.0 rakbit gm

- - - - - - - - -

* * * * * * * * * * * * *

FIGURE 2 Alignment of the predicted amino acid sequence of mouse glutaredoxin with other mammalians glutaredoxins. The homology values are calculated with respect to the mouse one. Asterisks indicate identical residues in all the mammalian pro- teins. The active site is boxed

codon at position 380 and codes for a protein of 107 amino acids with an estimated molecular weight of 11.9 kDa and a PI of 8.4. The mouse GRX protein displays a high degree of homology with the other mammalian glutaredoxins (Figure 2), including the conserved active site (except for the bovine protein in which the tyro- sine residue is changed for a phenylalanine). The availability of the mouse grx cDNA will be of great help for the isolation of its genomic sequence and transgenic / knock-out studies to elucidate further roles of glutaredoxin.

References Aslund, F., Ehn, B., Miranda-Vizuete, A,, Pueyo, C. and Hol-

mgren A. (1994) ”Two additional glutaredoxins exist in Esclierichin c d i : glutaredoxin-3 is a hydrogen donor in a thioredoxiniglutarcdoxin-1 double mutant”, Proc-. Nritl. Acnd. Sci. USA, 91,9813-9817.

Besse, I. and Buchanan, B.B. (1997) ”Thioredoxin-linked plant and animal processes: the new generation”, BotriJii- cnl Bulletin ofAcnderiiia Siriicri fTniLJei), 38, 1-1 1.

Hall, D.E., Baldesten, A., Holmgren, A. and Reichard, P. (1971) “Yeast thioredoxin. Amino-acid sequence around the active-center disulfide of thioredoxin I and [I” , E i i r . 1. Biocheiii., 23, 93-97.

Holmgren, A. (1979) ”Glutathione dependent synthesis of deoxyribonucleotides. Purification and characterization of glutaredoxin from Esc/iericliin coli”, I. B i d . C h i . , 254, 36643671.

Holmgren, A. (1989) “Thioredoxin and glutaredoxin sys- Acknowledgements This work was supported by grants from the Laurent, T.C., Moore, E.C. and Reichard, I‘. (1964) “ E n q Swedish Medical Research Council (Project matic synthesis of deoxyribonucleotides. isolation and

characterization of thioredoxin, the hydrogen donor from E~clicrichin coli B”, \. B i d . UZUJI. , 239, 3436-3444.

ska Institutet and the TMR Marie ‘IJrie Research Miranda-Vizuete, A,, Damdimopoulos, A.E., Gustafsson, Training Grants (Contract ERBFMBICT972824). J.-A. and Spyrou, C. (1997) “Cloning, expression and

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Padilla, C.A., Martinez-Galisteo, E., Bircena, J.A., Spyrou, G. and Holmgren, A. (1995) “Purification from placenta, amino acid sequence, structure comparisons and cDNA cloning of human glutaredoxin”, Eur. 1. Biochem., 227,

Pedrajas, J.R., Kosmidou, E., Miranda-Vizuete, A., Gustafs- son, J.-A., Wright, A.P.H. and Spyrou, G. (1998) ”Identi- fication and functional characterization of a novel mitochondria1 thioredoxin system in Sncchnrornycrs cere- stsine”, 1. Biol. Chem., in press.

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Spyrou, G., Enmark, E., Miranda-Vizuete, A. and Gustafsson, J.-A. (1997) ”Cloning and expression of a novel mamma- lian thioredoxin”, J . Biol. Chem., 272,2936-2941.

Tsang, M.L.S. (1981) “Asirnilatory sulfate reduction in Escherichin coli : identification of the alternate cofactor for adenosine 3’-phosphate 5‘-phosphosulfate reductase as glutaredoxin”, J. Bncteriol., 146,1059-1066.

Yang, Y., Gan, Z.-R. arid Wells W.W. (1989) ”Cloning and sequencing the cDNA encoding pig liver thioltrans- ferase”, Gene, 83,339-346.

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