Gene and Protein Characterization of the Human Glutathione S-Transferase Kappa and Evidence for a Peroxisomal Localization

Abstract

Kappa class glutathione S-transferase (GST) cDNA sequences have been identified in rat, mouse, and human. In the present study, we determined the structure and chromosomal location of the human GST Kappa 1 (hGSTK1) gene, characterized the protein, and demonstrated its subcellular localization. The human gene spans ∼5 kb, has 8 exons, and maps onto chromosome 7q34. The 5′-flanking region lacks TATA or CCAAT boxes, but there is an initiator element overlapping the transcription start site. hGSTK1 amino acid sequence showed homology to bacterial 2-hydroxychromene-2-carboxylate isomerase, an enzyme involved in naphthalene degradation pathway. hGSTK1 mRNA was expressed in all of the organs examined. Subcellular fractionation of HepG2 cells showed that the protein was located in peroxisomes and mitochondria and was not detectable in cytoplasm. The peroxisomal localization was confirmed by transfection of HepG2 cells with a plasmid coding a green fluorescent protein fused inframe to the N terminus of hGSTK1. The C terminus of hGSTK1 was essential for localization of the protein to peroxisomes, and the C-terminal sequence Ala-Arg-Leu represents a peroxisome targeting signal. This is the first time that a human GST has been found in peroxisomes, suggesting a new function for this family of enzymes. Kappa class glutathione S-transferase (GST) cDNA sequences have been identified in rat, mouse, and human. In the present study, we determined the structure and chromosomal location of the human GST Kappa 1 (hGSTK1) gene, characterized the protein, and demonstrated its subcellular localization. The human gene spans ∼5 kb, has 8 exons, and maps onto chromosome 7q34. The 5′-flanking region lacks TATA or CCAAT boxes, but there is an initiator element overlapping the transcription start site. hGSTK1 amino acid sequence showed homology to bacterial 2-hydroxychromene-2-carboxylate isomerase, an enzyme involved in naphthalene degradation pathway. hGSTK1 mRNA was expressed in all of the organs examined. Subcellular fractionation of HepG2 cells showed that the protein was located in peroxisomes and mitochondria and was not detectable in cytoplasm. The peroxisomal localization was confirmed by transfection of HepG2 cells with a plasmid coding a green fluorescent protein fused inframe to the N terminus of hGSTK1. The C terminus of hGSTK1 was essential for localization of the protein to peroxisomes, and the C-terminal sequence Ala-Arg-Leu represents a peroxisome targeting signal. This is the first time that a human GST has been found in peroxisomes, suggesting a new function for this family of enzymes. Glutathione S-transferases (GSTs) 1The abbreviations used are: GST, glutathione S-transferase; CBP, calmodulin-binding protein; DSBA, protein disulfide bond isomerase; EGFP, enhanced green fluorescent protein; HCCA, 2-hydroxychromene-2-carboxylate; PTS, peroxisomal targeting signal; 5′-RACE, rapid amplification of 5′ cDNA ends. are a family of ubiquitous intracellular enzymes that catalyze the conjugation of glutathione to many exogenous and endogenous compounds (1Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google Scholar). These include chemical carcinogens, therapeutic drugs, and products of oxidative stress. In addition to their major role in catalyzing the conjugation of electrophilic substrates to glutathione, these enzymes have GSH-dependent peroxidase (2Hurst R. Bao Y. Jemth P. Mannervik B. Williamson G. Biochem. J. 1998; 332: 97-100Crossref PubMed Scopus (128) Google Scholar) and isomerase (3Johansson A.S. Mannervik B. J. Biol. Chem. 2002; 277: 16648-16654Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) activities. GSTs also have a noncatalytic role via their interaction with kinases (4Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (966) Google Scholar, 5Gilot D. Loyer P. Corlu A. Glaise D. Lagadic-Gossmann D. Atfi A. Morel F. Ichijo H. Guguen-Guillouzo C. J. Biol. Chem. 2002; 277: 49220-49229Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 6Cho S.G. Lee Y.H. Park H.S. Ryoo K. Kang K.W. Park J. Eom S.J. Kim M.J. Chang T.S. Choi S.Y. Shim J. Kim Y. Dong M.S. Lee M.J. Kim S.G. Ichijo H. Choi E.J. J. Biol. Chem. 2001; 276: 12749-12755Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar) and their binding properties toward a wide range of endogenous and exogenous ligands (7Oakley A.J. Lo Bello M. Nuccetelli M. Mazzetti A.P. Parker M.W. J. Mol. Biol. 1999; 291: 913-926Crossref PubMed Scopus (113) Google Scholar, 8Oakley A.J. Lo Bello M. Mazzetti A.P. Federici G. Parker M.W. FEBS Lett. 1997; 419: 32-36Crossref PubMed Scopus (53) Google Scholar). Based primarily on protein sequence similarity, the soluble GSTs have been divided in eight families that are highly conserved between species: Alpha, Mu, Pi, Theta, Kappa, Sigma, Zeta, and Omega (1Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google Scholar, 9Pemble S.E. Wardle A.F. Taylor J.B. Biochem. J. 1996; 319: 749-754Crossref PubMed Scopus (265) Google Scholar, 10Blackburn A.C. Woollatt E. Sutherland G.R. Board P.G. Cytogenet. Cell. Genet. 1998; 83: 109-114Crossref PubMed Scopus (72) Google Scholar, 11Board P.G. Coggan M. Chelvanayagam G. Easteal S. Jermiin L.S. Schulte G.K. Danley D.E. Hoth L.R. Griffor M.C. Kamath A.V. Rosner M.H. Chrunyk B.A. Perregaux D.E. Gabel C.A. Geoghegan K.F. Pandit J. J. Biol. Chem. 2000; 275: 24798-24806Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar, 12Kanaoka Y. Fujimori K. Kikuno R. Sakaguchi Y. Urade Y. Hayaishi O. Eur. J. Biochem. 2000; 267: 3315-3322Crossref PubMed Scopus (82) Google Scholar). The GSTs of each family have short regions of high identity in common and share a common evolutionary pathway (13Sheehan D. Meade G. Foley V.M. Dowd C.A. Biochem. J. 2001; 360: 1-16Crossref PubMed Scopus (1431) Google Scholar). Soluble GSTs have been described mainly in cytoplasm but are also present in the nucleus and the mitochondrion where they could have functions peculiar to the integrity of these organelles (14Soboll S. Grundel S. Harris J. Kolb-Bachofen V. Ketterer B. Sies H. Biochem. J. 1995; 311: 889-894Crossref PubMed Scopus (65) Google Scholar, 15Raza H. Robin M.A. Fang J.K. Avadhani N.G. Biochem. J. 2002; 366: 45-55Crossref PubMed Google Scholar, 16Desmots F. Rissel M. Gilot D. Lagadic-Gossmann D. Morel F. Guguen-Guillouzo C. Guillouzo A. Loyer P. J. Biol. Chem. 2002; 277: 17892-17900Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). For example, GSTP1 in the nucleus might be associated with protection of DNA damage induced by doxorubicin and consequently in the acquisition of resistance to anticancer drugs by cancer cells (17Goto S. Ihara Y. Urata Y. Izumi S. Abe K. Koji T. Kondo T. FASEB J. 2001; 15: 2702-2714Crossref PubMed Scopus (102) Google Scholar), and the GSTs known to be present in the mitochondrion (i.e., GSTA1, GSTA4 and GSTM1) may play an important role in defense against chemical and oxidative stress (15Raza H. Robin M.A. Fang J.K. Avadhani N.G. Biochem. J. 2002; 366: 45-55Crossref PubMed Google Scholar). Among the eight classes of soluble GSTs, the Kappa class has been the least studied. In 1991, Harris et al. (18Harris J.M. Meyer D.J. Coles B. Ketterer B. Biochem. J. 1991; 278: 137-141Crossref PubMed Scopus (99) Google Scholar) reported the purification of a GST isolated from mitochondrial matrix of rat liver, GST13-13. Although this subunit was initially classified as a class Theta GST on the basis of N-terminal analysis, elucidation of its complete amino acid sequence by Pemble et al. (9Pemble S.E. Wardle A.F. Taylor J.B. Biochem. J. 1996; 319: 749-754Crossref PubMed Scopus (265) Google Scholar) showed the absence of sequence similarity to any other class of GST. Pemble et al. also identified human sequences in expressed sequence tag database that showed a high nucleotide similarity (77%) to that of rat GST subunit 13. Therefore, these GSTs were assigned to the Kappa class, with the rat GST subunit 13 and the human gene being rGSTK1 and hGSTK1, respectively. More recently, Jowsey et al. (19Jowsey I.R. Thomson R.E. Orton T.C. Elcombe C.R. Hayes J.D. Biochem. J. 2003; 373: 559-569Crossref PubMed Google Scholar) identified and characterized a murine class Kappa subunit: mGSTK1. This protein has been reported to be present at substantially higher levels in the mitochondrial fraction than the cytosolic fraction of rodent liver. The aim of the present study was to characterize the human GST Kappa at the gene and protein levels. Our results demonstrate that the hGSTK1 is encoded by a gene present on chromosome 7q34 and is expressed in all tissues studied, and its amino acid sequence presents similarities with a bacterial enzyme, 2-hydroxychromene-2-carboxylate (HCCA) isomerase. Furthermore, we showed for the first time the localization of a GST in the peroxisome, because hGSTK1 is present in this organelle. Materials—Rabbit anti-catalase antibody was a generous gift of Professor Völkl (Heidelberg, Germany). Rabbit anti-cytochrome c and caspase 3 were from Santa Cruz Biotechnology (Santa Cruz, CA). pDsRed2-peroxi and pEGFP-C1 plasmids were from Clontech Laboratories (Palo Alto, CA). Iodixanol was purchased as Optiprep from Axis-Shield (Oslo, Norway). In Silico Analysis—Nucleotide sequences of human GST Kappa 1 (GSTK1; GenBank™ accession number NM_015917) were compared with the High Throughput Genome Sequences subset of GenBank™ using BLASTN program with default parameters (NCBI; www.ncbi.nlm.nih.gov.BLAST). Comparative sequence alignments were generated using PipMaker (bio.cse.psu.edu). Repeat elements were characterized using RepeatMasker (ftp.genome.washington.edu/cgi-bin/RepeatMasker). Multiple alignments of protein sequences were performed using ClustalW (20Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar) and Boxshade. PCR—Amplification of genomic DNA was carried out using Platinum Taq DNA polymerase High Fidelity (Invitrogen). 5′-RACE—A modified 5′-RACE protocol that selects for 5′-capped mRNAs (FirstChoice™ RLM-RACE kit; Ambion, Austin, TX) was used to define the transcription initiation site(s) for hGSTK1 using the protocol recommended by the manufacturer. Briefly, cDNAs from human heart were amplified with nested primers to the RNA adapter (5′-GCTGATGGCGATGAATGAACACTG-3′) and an antisense hGSTK1-specific primer localized in exon 3 (5′-CATCACAGACAAGAAATCCT-3′). The PCR product was reamplified using a nested inner RNA adapter primer (Ambion, RLM-RACE kit) and an antisense hGSTK1-specific primer in exon 2 (5′-CCACTGTCTTTCATGATCCC-3′). PCR was carried out using Platinum Taq DNA polymerase High Fidelity (Invitrogen), using the at for 3 amplification for for for and at for PCR products from the amplification were on and were by DNA the the hGSTK1 cDNA sequence was amplified by PCR with Platinum Taq DNA polymerase High Fidelity using the sequences and for and respectively. was by using the primer and a primer the coding for the C-terminal The PCR were in the and of a protein of the enhanced protein with hGSTK1 to its C The were by DNA is a that a fluorescent protein peroxisomal targeting 1 and of hGSTK1 hGSTK1 was by using a and protein purification to hGSTK1 protein an N-terminal acid calmodulin-binding was in the by 1 for at The purification the of the with The cells were and in 2 1 1 was and the was with on for The cells were by and at for at The was on a in The was with of and of 2 1 1 The were in 2 of 1 were and by The hGSTK1 was with 1 of of The protein was for at against each of 2 of 1 and 1 against hGSTK1 protein was in using a protocol DNA was performed from the 5′ and of the genomic products using and an RNA and RNA was purchased from Clontech Laboratories (Palo RNA isolated from human The RNA from these tissues been from of each with the of heart and PCR were cDNA using the with the 1 of RNA and primers were used in each transcription cDNAs were at as in time PCR was performed by the fluorescent using the for from and the for hGSTK1 were with the 3 and Briefly, cDNA was with of of each enzyme, and of in a of first of at was by of amplification for and for For each the an amplification by the to the of to the was as the number at a in the was first The of the of the mRNA levels was of the of cDNA by an S. Subcellular cells were in with using a and The and mitochondria were by a at mitochondrial peroxisomes, and was by of the at for The peroxisomes were from the mitochondrial fraction by for in a of the protocol eight from the were with a by were in and of protein were on a and onto the were in they were with anti-cytochrome c or 3 The were in and and with peroxidase of the were at for 2 was using and cells were with the and of HepG2 cells was by with a from an to V. The cells were out and in with these the cells were in with were on an with a and using of the GST enzyme were at and with a In all the was and from the toward and and were at For product was determined using the of the glutathione product where the peroxidase was were as acid and The protein were by the of Biochem. PubMed Scopus Google Scholar). of the and of human GST Kappa cDNA sequence accession number NM_015917) to the High Throughput Genome Sequences subset of GenBank™ using the BLASTN program identified accession number located on chromosome 7q34. The sequence of this was as of a and between the and the Genome of cDNA with genomic sequences that the hGSTK1 gene spans and eight by and This genomic structure was confirmed by using human genomic DNA and amplification of each by PCR with primers localized in DNA was The nucleotide sequence in the GenBank™ nucleotide sequence the accession number The for the are for all exons, and an is present in exon 8 the region of the The at of the is of a found hGSTK1 gene structure with the described and rat GST Kappa (19Jowsey I.R. Thomson R.E. Orton T.C. Elcombe C.R. Hayes J.D. Biochem. J. 2003; 373: 559-569Crossref PubMed Google Scholar), we the sequences by the This program the identity in the the of regions the gene sequence 2 from exon This a high of genomic between the The of in these and is that the human sequence is than the rat and in In there is homology in the 5′-flanking regions between human and rodent sequence of The sequences for transcription The initiator sequence the transcription start as are in and sequences are in acid sequences are using the 5′-RACE PCR was performed to the transcription start site(s) of the human hGSTK1 using heart amplification of a was that the in 2 and localized of the could be the transcription start site. The region from the start CCAAT TATA but has a sequence to the initiator sequence overlapping the transcription initiation M.C. A.J. D. S. A. PubMed Scopus Google Scholar). and transcription and a region were also found in the of the in by and levels of hGSTK1 were by the time PCR hGSTK1 mRNA was expressed but at in all tissues and being in liver, and the hGSTK1 of amino acid sequence to the subset of GenBank™ using the program showed similarity to rat and GST Kappa, a GST and bacterial isomerase enzymes. of these sequences by using the and is in and respectively. The amino acid sequence of hGSTK1 is to that of rat and and with other identity was with C. GST and with and Furthermore, the protein family of alignments that the hGSTK1 amino acid sequence a conserved with DSBA, an involved in disulfide bond in the of similarities between amino acid sequences of hGSTK1 and E. or is than and of hGSTK1, a protein an N-terminal tag was in E. hGSTK1 was by of with The of and hGSTK1 were and as from toward and In toward and were not detectable of hGSTK1 substrates The are the of at least not detectable of in a new Subcellular of of hGSTK1 by the program a peroxisomal targeting at the C the and a for a mitochondrial at the N the of hGSTK1 in peroxisomes, we subcellular of HepG2 not we used of the mitochondrial This to their the of mitochondria of and peroxisomes of the major subcellular organelles with of subcellular of HepG2 cells using for hGSTK1, and caspase hGSTK1 was in peroxisomal and mitochondrial but not in and caspase 3 were in peroxisomes, mitochondria and respectively. the subcellular localization of hGSTK1 and the of the peroxisomal targeting at its C-terminal plasmid were with the cDNA the protein fused to the N terminus of hGSTK1 This was with that a of fluorescent protein and the peroxisomal targeting HepG2 the protein and the fluorescent peroxisomal protein were in peroxisomes and transfection of a the showed a of in nucleus and cytoplasm C and localization of in HepG2 with the and pDsRed2-peroxi HepG2 cells were and using of hGSTK1 in with in peroxisomes was confirmed by of and HepG2 cells with and The first Kappa class GST was described by Harris et al. (18Harris J.M. Meyer D.J. Coles B. Ketterer B. Biochem. J. 1991; 278: 137-141Crossref PubMed Scopus (99) Google Scholar), isolated from the rat mitochondrial a human cDNA was identified that a high of identity with rGSTK1 (9Pemble S.E. Wardle A.F. Taylor J.B. Biochem. J. 1996; 319: 749-754Crossref PubMed Scopus (265) Google Scholar). the hGSTK1 gene and protein have not been The gene hGSTK1 is located on chromosome 7q34. the for each GST class are on the chromosome M. L. A. Board P. Biochem. J. 1998; PubMed Scopus Google Scholar, F. C. Coles B. E. Guillouzo A. 2002; PubMed Scopus Google Scholar, S. Y. B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The that other sequences were found in this chromosomal region in other of the to that hGSTK1 is the in this GST family in The gene eight and spans The of the gene, its and the range of other human GST to The coding region of the hGSTK1 gene homology with and rat Kappa class subunit and the amino acid sequence and identity with and respectively. In there is a high of nucleotide sequence identity not in but also in between the rat, and The human cDNA sequence is also highly to expressed sequence identified in other species: and C. not hGSTK1 mRNA was found to be being found in all human tissues and the of Jowsey et al. (19Jowsey I.R. Thomson R.E. Orton T.C. Elcombe C.R. Hayes J.D. Biochem. J. 2003; 373: 559-569Crossref PubMed Google Scholar) of and rGSTK1 in eight The gene sequence and the range of of hGSTK1, with many other GSTs that of that hGSTK1 a role in of the 5′-flanking region of the hGSTK1 gene showed that there are similarities between the 5′-flanking sequences of human and rodent and suggesting between human and rodent gene was by the transcription start site. using human heart RNA and 5′-RACE we localized the transcription start the of the 5′-flanking region of this that the hGSTK1 gene lacks a TATA and a sequence at the a sequence present the transcription start has similarity to an initiator This initiator sequence has been to function as an in a TATA M.C. A.J. D. S. A. PubMed Scopus Google Scholar). is important for the initiator is present to of the transcription start site. the gene has been to an initiator element involved in its F. C. C. Guillouzo A. Morel F. Biochem. J. 1998; PubMed Scopus Google Scholar). the basis of its amino acid hGSTK1 is as of the is a of the family H. F. J. Rev. Biochem. 2003; PubMed Scopus Google Scholar). The and of bacterial in is a class of catalyze disulfide bond the of H. F. J. Rev. Biochem. 2003; PubMed Scopus Google Scholar). This family a of with a structure and is by an a by amino and by a in the structure of the 1 of E. The also isomerase catalyze in K.D. J. PubMed Scopus Google Scholar, J. 1994; PubMed Google Scholar, G. J. 1999; PubMed Google Scholar). showed that share the sequence similarity to Kappa class are involved in naphthalene degradation and have been from and and is that isomerase glutathione as a to to T. T. K. K. T. J. Biochem. 1998; PubMed Scopus Google Scholar). of amino acid sequence hGSTK1 from the other of the GST of the structure demonstrated N-terminal and C-terminal and not is to the but from the GST with an N-terminal and a C-terminal of (13Sheehan D. Meade G. Foley V.M. Dowd C.A. Biochem. J. 2001; 360: 1-16Crossref PubMed Scopus (1431) Google Scholar). Furthermore, the sequence lacks the located amino from the N terminus and found in all other classes of soluble hGSTK1 not to glutathione not similarities are found between hGSTK1 and other Although the structure from other GST using as a that hGSTK1 a to the G. H. and P. G. Biochem. in not This of hGSTK1 is confirmed by the structure of the rat GST Kappa in with glutathione PubMed Scopus Google Scholar). The of and the that the protein have from a common but by PubMed Scopus Google Scholar). The also that the Kappa class represents a protein that GST also its is toward substrates as and rGSTK1 was isolated from the matrix of mitochondria (18Harris J.M. Meyer D.J. Coles B. Ketterer B. Biochem. J. 1991; 278: 137-141Crossref PubMed Scopus (99) Google Scholar), we the subcellular of the human class Kappa GST. of amino acid sequence of hGSTK1 by the program a at the C-terminal this we used with on HepG2 subcellular by and transfection of HepG2 cells with fluorescent showed the of hGSTK1 in peroxisomal and mitochondrial was in of HepG2 cells with a plasmid the cDNA the fused to the N-terminal of hGSTK1 an fluorescent in transfection of a coding for the protein the N-terminal showed a of in nucleus and cytoplasm. This the of the peroxisomal for the of hGSTK1 from cytoplasm to This is in with peroxisomal These are on and the targeting to peroxisomes is by targeting sequences C. R. FEBS Lett. 2001; PubMed Scopus Google Scholar). of targeting sequences have been characterized for peroxisomal matrix and is the C-terminal or of its as C. R. FEBS Lett. 2001; PubMed Scopus Google Scholar). this peroxisomal targeting sequence is found in Kappa GST from other (i.e., rat, mouse, and C. suggesting that the of this GST in peroxisomes is conserved between The peroxisomal localization of rat and has not been described in the of in the mitochondrion has been demonstrated by using not the complete of peroxisomes and the of rGSTK1 and from mitochondrial (19Jowsey I.R. Thomson R.E. Orton T.C. Elcombe C.R. Hayes J.D. Biochem. J. 2003; 373: 559-569Crossref PubMed Google Scholar) not their in performed on rat peroxisomes and human heart the of a in rat peroxisomes M. S. T. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), a was found in human heart mitochondria E. B. D.E. S. Gibson 2003; PubMed Scopus Google Scholar). these that GST Kappa is present in mitochondria and The subcellular location of to its and peroxisomal functions are to and of that and R.J. Biochem. 2000; PubMed Scopus Google Scholar). study has demonstrated binding properties of class GSTs with and C. G. R. T. O. M. Eur. J. Biochem. 1999; PubMed Scopus Google Scholar). could be that the class Kappa GSTs are also involved in binding and play a role in the of or important of peroxisomes is that they the in the where and are generated C.J. Biochem. 1998; PubMed Scopus Google Scholar). Although peroxisomes enzymes and involved in the of present a major of and the on and Our results demonstrate a peroxidase of hGSTK1 toward and Therefore, GST Kappa might have an important role in of generated in these might also be found in mitochondria that are involved in and In study the and the of the human class Kappa GST. The similarities of hGSTK1 with enzymes that the Kappa class GSTs are an product of The of gene sequence and the ubiquitous of the Kappa class GSTs in tissues that the have a role in Furthermore, GST Kappa is the first human GST known to be localized in peroxisomes, is not in suggesting a new function for this family of enzymes. for