Isolation and chromosomal localization of the human glutathione peroxidase gene

We have isolated cDNA clones for the gene, termed GPX1, encoding the major human selenoprotein, glutathione peroxidase. Sequence analysis confirmed previous findings that the unusual amino acid seleno-cysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPX1 cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPX1 gene to a single site on chromosome 3, at region 3q11-13.1. Thus, three genomic sites bear sequence homology to the GPX1 cDNA, and the one most homologous maps to 3q11-13.1.     

Identification and localization of multiple forms of serine hydroxymethyltransferase in pea (Pisum sativum) and characterization of a cDNA encoding a mitochondrial isoform.

Serine hydroxymethyltransferase (SHMT) has been purified from the mitochondria of green pea leaves. Activity can be fractionated into two distinct peaks by ion exchange chromatography. While these two forms of the enzyme are immunologically indistinguishable, immunoinhibition experiments show the presence of a distinct non-mitochondrial third form of the enzyme to also be present in green pea leaves. While this mitochondrial form of SHMT is abundant in leaves it is absent from roots, although the two tissues have comparable SHMT activity. An antibody raised to purified mitochondrial SHMT was used to screen a cDNA expression library. The sequence of one of the isolated positive clones contained an open reading frame, which encoded a sequence that matched the amino acid sequence determined from the N terminus of the mature protein. The open reading frame encodes a mature protein of 487 amino acids with a M(r) of 54,000, together with a 27-31 amino acid serine-rich leader sequence, presumably required for mitochondrial targeting. The cDNA hybridizes to a small multigene family of 2-3 genes, which appear to be expressed predominantly in leaves. Comparison of the deduced amino acid sequence with the amino acid sequences of the rabbit mitochondrial and cytoplasmic SHMT, show that pea mitochondrial SHMT is equally similar to both of these enzymes. In addition, the rabbit sequences are more like one another than they are to the pea sequence, suggesting an interesting evolutionary relationship for these proteins.     

Molecular characterization of glutathione peroxidase-like protein in halotolerant Chlamydomonas sp. W80

A cDNA clone encoding a glutathione peroxidase (GPX)-like protein was isolated from the cDNA library from halotolerant Chlamydomonas W80 ( C . W80) by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 163 amino acids with a calculated molecular mass of 18 267 Da. No potential signal peptide was found. The deduced amino acid sequence of the cDNA showed 40–63% and 37–46% homology to those of GPX-like proteins from higher plants and mammalian GPXs, respectively. The C. W80 GPX-like protein contained a normal cysteine residue instead of a selenocysteine at the catalytic site. However, it contained amino acid residues (glutamine and tryptophan) that are involved in three protein loops and are important for the catalytic activity in the mammalian GPX. Interestingly, the native and recombinant GPX-like proteins showed activities towards unsaturated fatty acid hydroperoxides, but not towards either H₂O₂ or phospholipid hydroperoxide. Transformed E. coli cells expressing the C . W80 GPX-like protein showed enhanced tolerance to 5% NaCl or 0.2 m M paraquat treatments.
Accession number : The nucleotide sequence data reported have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases with the following accession number AB009083.     

Molecular cloning of cDNA coding for rat plasma glutathione peroxidase

The plasma glutathione peroxidase (PGSH-PO), which is different from erythrocyte glutathione peroxidase (EGSH-PO) in immunochemical property and substrate specificity, was purified from male Wistar rat serum. The amino acid sequence of 5 independent peptides were determined and a cDNA clone for this enzyme was isolated from placental cDNA library. The nucleotide sequence of the cDNA revealed that, similar to EGSH-PO cDNA, the seleno-cysteine was genetically encoded by "TGA" codon. On comparing the nucleotide sequences of EGSH-PO and PGSH-PO, no significant homology was found in the vicinities of "TGA" codons of both enzymes.     

Cloning and sequence analysis of the human liver rhodanese: comparison with the bovine and chicken enzymes

The cDNA for the human rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), a nuclearly encoded protein of the mitochondrial matrix, was isolated from a human fetal liver cDNA library. Nucleotide sequence revealed an open reading frame coding for a polypeptide of 295 amino acids, which presented a 57% and 58% identity with the bovine and avian rhodanese, respectively. The analysis of the 5'-ends of the coding region gave no evidence for the presence of a cleavable signal sequence as found in other mitochondrial proteins. A comparison with two available amino acid sequences (cow and chicken) showed that sequence similarity is not restricted to the alpha-helices and beta-structures motifs which are remarkably superimposable in the two halves of bovine rhodanese, but extends to adjacent regions.     

Molecular characterization and mRNA expression of glutathione peroxidase and glutathione S-transferase during osmotic stress in olive flounder (Paralichthys olivaceus)

Glutathione peroxidase (GPX) and glutathione S-transferase (GST) are key enzymes of cellular detoxification systems that defend cells against reactive oxygen species (ROS). In this study, we isolated the GPX and GST full-length cDNA and investigated the expression of these mRNAs from livers of olive flounder during salinity changes (35, 17.5, 8.75, 4 and 0 psu) by quantitative PCR (QPCR). GPX cDNA consists of 429 base pairs (bp) and encodes a protein of 142 amino acids. GST cDNA consists of 663 bp and encodes a protein of 220 amino acids. Both of GPX and GST mRNA expressions were the highest in 4 psu and then decreased in 0 psu. Also, the levels of Na(+) and Cl(-) decreased, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased during the experimental period. These findings provide molecular characterization of GPX and GST in olive flounder and suggest that GPX and GST play important roles in detoxification of ROS, thereby these maybe indicators of oxidative stress responses by salinity changes in olive flounder.     

Isolation and characterization of a novel PHGPx gene in Raphanus sativus

A full-length cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase (PHGPx) was cloned from Raphanus sativus. The cDNA, designated RsPHGPx, includes an open reading frame which encodes 197 amino acid residues. The alignment of amino acid sequences showed that RsPHGPx had the highest sequence homology to plant PHGPx and contained an N-terminal extension characteristic of a mitochondrial targeting peptide. Northern blot analysis indicated that RsPHGPx was constitutively and ubiquitously expressed during radish development, and its expression was differently regulated by various stress conditions. The expression of RsPHGPx in a yeast PHGPx-deletion mutant significantly rescued the mutant sensitivity to oxidation-sensitive linolenic acid, just as the yeast PHGPx3 gene did. This suggested that RsPHGPx encodes a functional PHGPx protein.     

cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A 64 kD protein was enriched from rat liver mito-chondria during the purification of choline dehydro-genase (CHDH)[1]. Homologous comparison and func-tional experiments demonstrated that the protein was electron-transfer flavoprotein-ubiquinone oxidoreduc-tase protein (ETF-QO). The N-terminal sequence determination of rat liver ETF-QO protein purified by various methods did not provide unequivocal result. However, when the protein was digested with V8 protease, peptide fragments could b…     

A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast

The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the mitochondrial membrane potential, and alters the intracellular redox potential. Co-expression of the BI-GST/GPX protein brought the total glutathione levels back to normal and re-established the mitochondrial membrane potential but had no effect on the phospholipid alterations. Moreover, expression of BI-GST/GPX in yeast was found to significantly enhance resistance to H(2)O(2)-induced stress. These results underline the relationship between oxidative stress and Bax-induced death in yeast cells and demonstrate that the yeast-based genetic strategy described here is a powerful tool for the isolation of novel antioxidant and antiapoptotic genes.     

The structurally distinct form of pp60c-src detected in neuronal cells is encoded by a unique c-src mRNA.

A cellular src (c-src) cDNA clone was isolated from a chicken embryonic brain cDNA library and characterized by DNA sequence analysis. Comparison with the published sequence of a chicken genomic c-src clone indicated that the brain cDNA clone contained an 18-base-pair insertion located between exons 3 and 4 of the c-src gene. The six amino acids encoded by the insertion caused an alteration in the electrophoretic mobility of the c-src gene product similar to that of the structurally distinct form of the src protein detected in neuronal cultures.     

cDNA cloning,functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5′-terminal and 3′-terminal were obtained by RACE technique. The full-length cDNAthat encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-QOp. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.     

Molecular cloning of a cDNA encoding chicken T-protein of the glycine cleavage system and expression of the functional protein in Escherichia coli. Effect of mRNA secondary structure in the translational initiation region on expression.

DNA clones encoding chicken T-protein of the glycine cleavage system were isolated from chicken liver lambda gt10 cDNA libraries. Three overlapping clones provided an open reading frame of 1176 nucleotides that predicts a polypeptide of 392 amino acids (M(r) 42,056) comprised of a 16-residue mitochondrial targeting sequence and a 376-residue mature protein (M(r) 40,292). The amino acid sequence predicted for the mature protein showed 67% identity with that of bovine T-protein. A cDNA encoding mature T-protein was constructed, and the nucleotide sequence just downstream of the initiation codon was modified without amino acid substitution to reduce the free energy of formation for the folded mRNA. Expression plasmids containing these cDNA variants produced large amounts of T-protein in Escherichia coli, while very low expression was observed with a plasmid containing wild type cDNA. Enzymatically active T-protein was obtained when the expression was conducted at 30 degrees C with 25 microM isopropyl-1-thio-beta-D-galactopyranoside. Under the full inducing condition (at 37 degrees C and 1 mM inducer), the expressed T-protein was recovered as insoluble and inactive protein. The recombinant T-protein was purified to near homogeneity with a yield of about 30%. Apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is approximately 40,000, similar to the size of T-protein purified from chicken liver. NH2-terminal amino acid sequence analysis (9 residues) revealed 100% identity with chicken T-protein determined chemically. The kinetic properties of the recombinant T-protein resembled those of the native chicken T-protein.     

Identification of a hamster sperm 26-kilodalton dehydrogenase/reductase that is exclusively localized to the mitochondria of the flagellum

Sperm mitochondria undergo remodeling during posttesticular maturation that includes extensive disulfide cross-linking of proteins of the outer membrane to form the insoluble mitochondrial capsule. The relationship of these changes to mitochondrial function in mature gametes is unclear. The phospholipid hydroperoxide glutathione peroxidase (GPX4; also termed PHGPx) represents a major disulfide bond-stabilized protein of the mitochondrial capsule, and it is readily released by disulfide-reducing agents. However, in addition to GPX4, we detected a second major protein of 26 kDa (MP26) that was eluted from purified hamster sperm tails by the disulfide-reducing agent dithiothreitol. The objectives of the present study were to identify and characterize MP26 and to explore its potential role in mitochondrial function. Proteomic analysis of MP26 by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identified 14 peptides with sequence identity to a member of the short-chain dehydrogenase/reductase superfamily termed P26h, which was implicated previously in hamster sperm-zona binding, and with high sequence similarity to mouse lung carbonyl reductase. Indirect immunofluorescence localized MP26 to the midpiece, and two-dimensional PAGE and immunoblot analysis identified a single MP26 isoform of pI 9.0. Immunoblot analyses of cauda epididymal fluid and of purified sperm plasma membranes and mitochondria revealed the exclusive localization of MP26 to the mitochondrial fraction. These data indicate that MP26 does not function in zona binding; instead, like GPX4, it may be associated with the mitochondrial capsule and play an important role in sperm mitochondrial function.     

The cDNA sequence of a neutral horseradish peroxidase

A cDNA clone encoding a horseradish (Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic pI value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition with the HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.