The selenoenzyme phospholipid hydroperoxide glutathione peroxidase

The reduction of membrane-bound hydroperoxides is a major factor acting against lipid peroxidation in living systems. This paper presents the characterization of the previously described 'peroxidation-inhibiting protein' as a 'phospholipid hydroperoxide glutathione peroxidase'. The enzyme is a monomer of 23 kDa (SDS-polyacrylamide gel electrophoresis). It contains one gatom Se/22 000 g protein. Se is in the selenol form, as indicated by the inactivation experiments in the presence of iodoacetate under reducing conditions. The glutathione peroxidase activity is essentially the same on different phospholipids enzymatically hydroperoxidized by the use of soybean lipoxidase (EC 1.13.11.12) in the presence of deoxycholate. The kinetic data are compatible with a tert-uni ping-pong mechanism, as in the case of the 'classical' glutathione peroxidase (EC 1.11.1.9). The second-order rate constants (K1) for the reaction of the enzyme with the hydroperoxide substrates indicate that, while H2O2 is reduced faster by the glutathione peroxidase, linoleic acid hydroperoxide is reduced faster by the present enzyme. Moreover, the phospholipid hydroperoxides are reduced only by the latter. The dramatic stimulation exerted by Triton X-100 on the reduction of the phospholipid hydroperoxides suggests that this enzyme has an 'interfacial' character. The similarity of amino acid composition, Se content and kinetic mechanism, relative to the difference in substrate specificity, indicates that the two enzymes 'classical' glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are in some way related. The latter is apparently specialized for lipophylic, interfacial substrates.     

The role of phospholipid hydroperoxide glutathione peroxidase isoforms in murine embryogenesis

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice.     

谷胱甘肽磷脂氢过氧化物酶研究进展

谷胱甘肽磷脂氢过氧化物酶(PHGPx)是生物体内一种重要的抗氧化酶。它是一种硒依赖性蛋白,在谷胱甘肽(GSH)的参与下能特异性地还原磷脂氢过氧化物(PLOOH)和胆固醇氢过氧化物(ChOOH),从而保护生物膜免受过氧化损伤。它还是核酸等生物大分子的重要保护剂,并且在细胞凋亡调控中发挥作用。     

Testis glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase activities in aminoguanidine-treated diabetic rats

Severe steroidogenic and spermatogenic alterations are reported in association with diabetic manifestations in humans and experimental animals. This study was planned to determine whether oxidative stress is involved in diabetes-induced alterations in the testes. Diabetes was induced in male rats by injection of 50 mg/kg of streptozotocin (STZ). Ten weeks after injection of STZ, levels of selenium and activities of selenium dependent-glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) were measured in rat testis. Lipid and protein oxidations were evaluated as measurements of testis malondialdehyde (MDA) and protein carbonyl levels, respectively. Testis sulfydryl (SH) levels were also determined. The control levels of GPx and PHGPx activities were found to be 46.5 +/- 6.2 and 108.8 +/- 19.8 nmol GSH/mg protein/min, respectively. Diabetes caused an increase in testis GPx (65.0 +/- 21.1) and PHGPx (155.9 +/- 43.1) activities but did not affect the levels of selenium or SH. However, the testis MDA and protein carbonyl levels as markers of lipid and protein oxidation, respectively, did not increase in the diabetic group. Aminoguanidine (AG) treatment of diabetic rats returned the testis PHGPx activity (136.5 +/- 24.9) to the control level but did not change the value of GPx activity (69.2 +/- 17.4) compared with diabetic group. MDA and protein carbonyl levels in testis were not affected by AG treatment of diabetic rats, but interestingly AG caused SH levels to increase. The results indicate that reactive oxygen radicals were not involved in possible testicular complications of diabetes because diabetes-induced activations of GPx and PHGPx provided protection against oxidative stress, which was reported to be related to some diabetic complications.     

Embryonic expression profile of phospholipid hydroperoxide glutathione peroxidase

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is indispensable for murine embryonic development; yet, the cellular mechanisms leading to embryonic death around gastrulation are still unclear. To investigate PHGPx expression patterns during embryogenesis, we performed a detailed analysis that revealed a complex expression profile. Up to embryonic day 9.5, PHGPx was ubiquitously expressed, which was, albeit to a lower extent, maintained throughout later stages of embryogenesis. Notably, strong expression was frequently observed in epithelial tissue. A transient increase in PHGPx expression was detected in developing tissues, suggesting a crucial role for PHGPx in proliferation and differentiation. By semi-quantitative RT-PCR analysis we observed that the cytosolic form of PHGPx was present in embryonic and somatic tissues whereas the mitochondrial and nuclear forms were detectable only in testicular tissue. This strongly suggests that it is the cytosolic form of PHGPx that is indispensable for embryonic development.     

Involvement of mitochondrial phospholipid hydroperoxide glutathione peroxidase as an antiapoptotic factor

Although reactive oxygen species (ROS) such as superoxide and hydroperoxide are known to induce apoptotic cell death, little is known as to the apoptotic death signaling of mitochondrial ROS. Recent evidence has suggested that antioxidant enzymes in mitochondria may be responsible for the regulation of cytochrome c release and apoptotic cell death. This paper examines the current state of knowledge regarding the role of mitochondrial antioxidant enzymes, especially phospholipid hydroperoxide glutathione peroxidase. A model for the release of cytochrome c by lipid hydroperoxide has also been proposed.     

Prevention of geranylgeranoic acid-induced apoptosis by phospholipid hydroperoxide glutathione peroxidase gene

Micromolar concentrations (0.5 approximately 5 microM) of all-trans geranylgeranoic acid (GGA) induced cell death in a guinea pig cell line, 104C1, whereas under the same conditions GGA was unable to kill 104C1/O4C, a clone established from 104C1 cells by transfection of them with the human phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene. GGA (5 microM) induced a loss of the mitochondrial inner membrane potential (DeltaPsim) in 104C1 cells in 2 h, and their apoptotic cell death became evident in 6 h. On the other hand, 104C1/O4C cells were resistant to loss of DeltaPsim and showed intact morphology until at least 24 h after addition of 10 microM GGA. Dihydroethidine, superoxide-sensitive probe, was immediately oxidized 15 min after addition of GGA in both 104C1 and 104C1/O4C cells. The peroxide-sensitive probe 2',7'-dichlorofluorescin diacetate (H2-DCF-DA) was strongly oxidized in 104C1 cells 4 h after the addition of 2.5 microM GGA, but not in 104C1/O4C cells even in the presence of 10 microM GGA. The present results suggest that GGA induced a hyper-production of superoxide and subsequently peroxides, which in turn may have led to dissipation of the DeltaPsim and final apoptotic cell death in 104C1 cells.     

Male fertility is linked to the selenoprotein phospholipid hydroperoxide glutathione peroxidase

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) accounts for almost the entire selenium content of mammalian testis. PHGPx is abundantly expressed in spermatids as active peroxidase but is transformed to an oxidatively inactivated protein in mature sperm, where it is a major constituent of the mitochondrial capsule in the midpiece. Male infertility in selenium-deficient animals, which is characterized by impaired sperm motility and morphological midpiece alterations, is considered to result from insufficient PHGPx content. We studied the relationship between sperm PHGPx, measured as rescued activity, and human fertility. Sperm specimens from 75 infertile men and 37 controls were analyzed for fertility-related parameters according to World Health Organization criteria. The PHGPx protein content was estimated after reductive solubilization of the spermatozoa by measuring the rescued PHGPx activity. Rescued PHGPx activity of infertile men ranged significantly below that of controls (93.2 +/- 60.1 units/mg sperm protein vs. 187.5 +/- 55.3 units/mg) and was particularly low in oligoasthenozoospermic specimens (61.93 +/- 45.42 units/mg; P < 0.001 compared with controls and asthenozoospermic samples). Rescued PHGPx activity was correlated positively with viability, morphological integrity, and most profoundly forward motility (r = 0.35, 0.44, and 0.45, respectively). In isolated motile samples, motility decreased faster with decreasing PHGPx content. In humans, PHGPx appears to be indispensable for structural integrity of spermatozoa and to codetermine sperm motility and viability. Because the content of PHGPx, irrespective of the cause of alteration, is correlated with fertility-related parameters, PHGPx can be considered a predictive measure for fertilization capacity.     

Involvement of phospholipid hydroperoxide glutathione peroxidase in the modulation of prostaglandin D2 synthesis

Antigenic cross-linking of the high affinity IgE receptors on mast cells induced the synthesis of prostaglandin D(2) (PGD(2)). The production of PGD(2) in L9 cells, which overexpressed non-mitochondrial phospholipid glutathione peroxidase (PHGPx), was only one-third that in the control line of cells (S1 cells). The reduction in the formation of PGD(2) in L9 cells was reversed upon inhibition of PHGPx activity by buthionine sulfoximine. Experiments with inhibitors demonstrated that prostaglandin H synthase-2 (PGHS-2) was the isozyme responsible for the production of PGD(2) upon cross-linking of IgE receptors. The conversion of radiolabeled arachidonic acid to prostaglandin H(2) (PGH(2)) was strongly inhibited in L9 cells, whereas the rate of conversion of PGH(2) to PGD(2) was the same in L9 cells and S1 cells, indicating that PGHS was inactivated in L9 cells. The PGHS activity in L9 cells was about half that in S1 cells. However, PGHS activity in L9 cells increased to the level in S1 cells upon the addition of the hydroperoxide 15-hydroperoxyeicosatetraenoic acid or of 3-chloroperoxybenzoic acid. These results suggest that non-mitochondrial PHGPx might be involved in the inactivation of PGHS-2 in nucleus and endoplasmic reticulum via reductions in levels of the hydroperoxides that are required for full activation of PGHS. Therefore, it appears that PHGPx might function as a modulator of the production of prostanoids, in addition to its role as an antioxidant enzyme.     

Molecular cloning and functional expression of nucleolar phospholipid hydroperoxide glutathione peroxidase in mammalian cells

We cloned a full-length cDNA for phospholipid hydroperoxide glutathione peroxidase (PHGPx) including exon Ib from rat and mouse testis. The nuclear signal sequence of the N terminal of rat nuclear PHGPx possessed a different sequence from that previously reported for rat sperm nuclei GPx (SnGPx). Expression of this PHGPx-YFP (yellow fluorescent protein) fusion protein including a novel nuclear signal sequence was exclusively localized in nucleolus; although YFPs fused with only a novel nuclear signal sequence were distributed in the whole nucleus, indicating that preferential translocation of nucleolar PHGPx into nucleoli was required for the nuclear signal sequence and internal sequence of PHGPx. Low level expression of nucleolar PHGPx was detected in several tissues, but the expression of nucleolar PHGPx was extensively high in testis. Immunohistochemical analysis with anti-nucleolar PHGPx indicated that expression of nucleolar PHGPx was observed in the nucleoli in the spermatogonia, spermatocyte, and spermatid. Overexpression of 34kDa nucleolar PHGPx in RBL2H3 cells significantly suppressed cell death induced by actinomycin D and doxorubicin that induced damage in the nucleolus. These results indicated that nucleolar PHGPx plays an important role in prevention of nucleolus from damage in mammalian cells.     

Molecular cloning and expression of a phospholipid hydroperoxide glutathione peroxidase homolog in Oryza sativa

A cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase (PHGPX) was isolated from rice using rapid amplification of cDNA ends. This cDNA, designated ricPHGPX, includes an open reading frame encoding a protein of 169 amino acids which shares about 60% and 50% amino acid sequence identity with plant and mammalian PHGPXs, respectively. The gene is expressed at a relative high level in flag leaves and the expression can be markedly induced by oxidative stress, suggesting that the product of the gene plays a key role in defense against oxidative damage in rice.     

Inhibitory effect of phospholipid hydroperoxide glutathione peroxidase on the activity of lipoxygenases and cyclooxygenases

The partially purified phospholipid hydroperoxide glutathione peroxidase (PHGPx) from A431 cells was used to systematically compare the inhibitory effect on the enzyme activity of various lipoxygenases and cyclooxygenases. Under the standard assay system, platelet 12-lipoxygenase, 15-lipoxygenase, and cyclooxygenase-2 were the most sensitive to the inhibition by PHGPx. 5-Lipoxygenase and cyclooxygenase-1 were less sensitive to the inhibition by PHGPx than platelet 12-lipoxygenase and cyclooxygenase-2, respectively, and the difference was approximately 10-fold. Reduction of 12(S)-hydroperoxyeicosatetraenoic acid to 12(S)-hydroxyeicosatetraenoic acid by PHGPx was observed in the presence of glutathione (GSH), and the inhibitory effect of PHGPx on 12-lipoxygenase-catalyzed arachidonate metabolism was reversed by the addition of exogenous lipid hydroperoxide. The results indicate that PHGPx directly reduced lipid hydroperoxides and then down-regulated the activity of arachidonate oxygenases. Moreover, a high-level expression of PHGPx mRNA and its 12-lipoxygenase-inhibitory activity was observed in cancer cells and endothelial cells, and these results suggest that PHGPx may play a significant role in the regulation of reactive oxygen species formation in these cells.     

Purification and physicochemical characterization of a recombinant phospholipid hydroperoxide glutathione peroxidase from Oryza sativa

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes. In the present work, the entire encoding region for Oryza sativa PHGPx was expressed in Escherichia coli M15, and the purified fusion protein showed a single band with 21.0 kD and pI = 8.5 on SDS- and IFE-PAGE, respectively. Judging from CD and fluorescence spectroscopy, this protein is considered to have a well-ordered structure with 12.2% alpha-helix, 30.7% beta-sheet, 18.5% gamma-turn, and 38.5% random coil. The optimum pH and temperature of the enzyme activity were pH 9.3 and 27 degrees C. The enzyme exhibited the highest affinity and catalytical efficiency to phospholipid hydroperoxide employing GSH or Trx as electron donor. Moreover, the protein displayed higher GSH-dependent activity towards t-Butyl-OOH and H(2)O(2). These results show that OsPHGPx is an enzyme with broad specificity for hydroperoxide substrates and yielded significant insight into the physicochemical properties and the dynamics of OsPHGPx.