The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the major molecular target of selenodeficiency in rodents, accounting for most of the histopathological and structural abnormalities of testicular tissue and male germ cells. PHGPx exists as a cytosolic form, mitochondrial form, and nuclear form (nPHGPx) predominantly expressed in late spermatids and spermatozoa. Here, we demonstrate that mice with a targeted deletion of the nPHGPx gene were, unlike mice with the full knockout (KO) of PHGPx, not only viable but also, surprisingly, fully fertile. While both morphological analysis of testis and epididymis and sperm parameter measurements did not show any apparent abnormality, toluidine blue and acridine orange stainings of spermatozoa indicated defective chromatin condensation in the KO sperm isolated from the caput epididymis. Furthermore, upon drying and hydrating, KO sperm exhibited a significant proportion of morphologically abnormal heads. Monobromobimane labeling and protein-free thiol titration revealed significantly less extensive oxidation in the cauda epididymis when compared to that in the wild type. We conclude that nPHGPx, by acting as a protein thiol peroxidase in vivo, contributes to the structural stability of sperm chromatin.

PHGPx and spermatogenesis.

PHGPx of rat sperm mitochondrial capsule is cross-linked and inactive. The enzyme is in part released in an active form by mercaptoethanol. Treatment with H(2)O(2) of reduced and solubilised capsule proteins, in the absence of any added reductant, results in: i) H(2)O(2) consumption which depends on the presence of both, PHGPx activity and protein thiols; ii) protein thiol oxidation with a stoichiometry of 2 equivalents of thiol per mole of hydroperoxide and, iii) PHGPx inactivation and cross-linking. SDS-PAGE analysis of monobromobimane-labeled proteins, following incubation with H(2)O(2), shows that the oxidation takes place in specific bands in the area of 20~kDa. It is concluded that the protein thiol peroxidase activity of PHGPx is responsible for cross-linking proteins in the mammalian sperm capsule and accounts for the selenium dependency of spermatogenesis.

Lipid peroxidation in human proteinuric disease.

BACKGROUND: While metabolically generated oxidants are produced locally in experimental glomerular diseases, little is still known of their significance and the respective scavenger systems in human glomerular diseases. METHODS: Here we studied kidneys from patients with congenital nephrotic syndrome of the Finnish type (CNF), a human model disease of isolated proteinuria. Expression of specific mRNAs for a major antioxidant system against lipoperoxidation [phospholipid hydroperoxide glutathione peroxidase (PHGPx)] and for mitochondrial proteins were studied in Northern blotting together with analysis of PHGPx in semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The respective proteins and lipoperoxide (LPO) adducts malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were analyzed in immunohistochemistry. RESULTS: PHGPx and the mitochondrially encoded subunits of cytochrome-c-oxidase were distinctly down-regulated within the glomeruli of CNF kidneys. These changes were confirmed in semiquantitative RT-PCR. Increases of lipoperoxidation products MDA and 4-HNE were constantly found in the glomeruli of CNF. In agreement with findings in CNF, similar results were obtained in biopsies from other human glomerular diseases. CONCLUSIONS: These findings suggest that local mitochondrial damage initiates LPO, which then causes deposition of the cytotoxic LPO products in glomeruli, as seen especially in CNF kidneys. Together with down-regulation of the local antioxidant protection, these may be important pathophysiologic mechanisms in human glomerular disease.

Dual function of the selenoprotein PHGPx during sperm maturation.

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) changes its physical characteristics and biological functions during sperm maturation. PHGPx exists as a soluble peroxidase in spermatids but persists in mature spermatozoa as an enzymatically inactive, oxidatively cross-linked, insoluble protein. In the midpiece of mature spermatozoa, PHGPx protein represents at least 50 percent of the capsule material that embeds the helix of mitochondria. The role of PHGPx as a structural protein may explain the mechanical instability of the mitochondrial midpiece that is observed in selenium deficiency.

Testosterone mediates expression of the selenoprotein PHGPx by induction of spermatogenesis and not by direct transcriptional gene activation.

Selenium deficiency is known to be associated with male infertility, and the selenoprotein PHGPx has been shown to increase in rat testis after puberty and to depend on gonadotropin stimulation in hypophysectomized rats [Roveri et al. (1992) J. Biol. Chem. 267, 6142 6146]. Exposure of decapsulated whole testis, however, failed to reveal any transcriptional activation or inhibition of the PHGPx gene by testosterone, human chorionic gonadotropin, or forskolin. Nevertheless, it was verified that the specific activity of PHGPx in testis, but not of cGPx, correlated with sexual maturation. Leydig cell destruction in vivo by ethane dimethane sulfonate (EDS) resulted in a delayed decrease in PHGPx activity and mRNA that could be completely prevented by testosterone substitution. cGPx transiently increased upon EDS treatment, probably as a result of reactive macrophage augmentation. In situ mRNA hybridization studies demonstrated an uncharacteristic low level of cGPx transcription in testis, whereas PHGPx mRNA was abundantly and preferentially expressed in round spermatids. The data show that the age or gonadotropin-dependent expression of PHGPx in testis does not result from direct transcriptional gene activation by testosterone, but is due to differentiation stage-specific expression in late spermatids, which are under the control of Leydig cell-derived testosterone. The striking burst of PHGPx expression at the transition of round to elongated spermatids suggests an involvement of this selenoprotein in sperm maturation.

Probing the presumed catalytic triad of a selenium-containing peroxidase by mutational analysis.

Glutathione peroxidases (GPx) are characterized by a catalytically active selenium which forms the center of a strictly conserved triad composed of selenocysteine, glutamine, and tryptophan. In order to check the functional relevance of this structural peculiarity, six molecular mutants of phospholipid hydroperoxide glutathione peroxidase (PHGPx) were designed, isolated, and investigated kinetically. Replacement of the selenocysteine in position 46 by cysteine decreased k + 1, i.e., the reaction rate of reduced enzyme with hydroperoxide, by three orders of magnitude. The rate of regeneration of the reduced enzyme by glutathione (k' + 2) was similarly affected. Additional substitution of Gln81 or Trp136 by acid residues resulted in a further decrease of k + 1 by three orders of magnitude, whereas histidine or neutral residues in these positions proved to be less deleterious. The data support the hypothesis that the typical triad of selenocysteine, glutamine, and tryptophan is indeed a novel catalytic center in which the reactivity of selenium is optimized by hydrogen bonding provided by the adjacent glutamine and tryptophan residues.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx): more than an antioxidant enzyme?

The family of glutathione peroxidases encompasses, as far, three tetrameric glutathione peroxidases (GPx) and the monomeric PHGPx. Although the overall homology between tetrameric enzymes and PHGPx is less than 30%, a pronounced similarity has been detected on clusters involved in the active site and a common catalytic triad (selenocysteine glutamine and tryptophan) has been defined by structural and kinetic data. A major peculiar feature of the reaction catalyzed by PHGPx is the possibility to accommodate large lipophilic substrates. This accounts for the observed dramatic antiperoxidant effect and the synergism with vitamin E. Moreover, the reduction of lipid hydroperoxides accounts also for the observed modulation of cycloxygenase and inhibition of 15-lipoxygenase. On the other hand, structural and kinetic data indicate that also the specificity of PHGPx for the donor substrate is not restricted to GSH and the recent observation the PHGPx binds to specific mitochondrial proteins, from which it is released by ionic strength and thiols, suggests a possible fole of this selenoenzyme in catalyzing the specific oxidation of protein thiols, thus modulating the activity of cellular regulatory elements. On this light, the selenium mojety of PHGPx, reacting much faster that thiols with a peroxide, and then oxidizing specific protein thiols, would channel the oxidation toward protein targets, thus providing, by protein-protein interaction, the specificity of the redox transition.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) in rat testis nuclei is bound to chromatin.

In rat testis nuclei the activity of the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx, EC 1.11.1.12) is much higher than in other tissues and subcellular compartments, with the sole exception of mitochondria. In nuclei, the bound enzyme is solubilized by DNase I treatment, thus suggesting a binding to chromatin. Treatment with ionic strength releases about 70% of bound PHGPx, suggesting that electrostatic bonds are involved. Immunogold electron microscopy indicates the association of PHGPx with chromatin structures in isolated nuclei. A possible interpretation of these data is a PHGPx protective role against DNA peroxidative damage. Furthermore, in agreement with kinetic and structural information, PHGPx-chromatin binding could suggest an hypothetical thiol oxidase activity toward specific thiol bearing proteins which could substitute for GSH as alternative donor substrates. Such activity could give to the enzyme a new important function which is not only protective but also has a specific regulatory function in chromatin condensation.

Probing the presumed catalytic triad of selenium-containing peroxidases by mutational analysis of phospholipid hydroperoxide glutathione peroxidase (PHGPx).

Single and double site mutants affecting the presumed catalytic centre of the selenoenzyme PHGPx were subjected to functional analysis. The rate constants k+1 and k'+2, for the oxidation and the regeneration of the ground state enzyme were estimated, respectively. Moreover, the alkylation rate of the reactive centre by iodoacetate (kinact.) was also analysed. The substitution of the catalytically competent selenocysteine 46 by cysteine (PHGPxcys46) decreased k+1 and k'+2 by about three orders of magnitude, although leaving unaffected kinact.. Furthermore, mutations of PHGPxcys46 involving the other residues of the triad decreased both kinact. and k+1, thus highlighting the involvement of Gln 81 and Trp 136 in the dissociation/activation of the nucleophilic cysteine thiol. In general, substitutions of Gln 81 or Trp 136 by acidic residues in PHGPxcys46 most dramatically depressed the k+1 values, because they practically prevented the dissociation of the thiol group, while neutral or positively charged residues in these positions allowed an intermediate dissociation and induced a corresponding reactivity of the thiol. Our data, for the first time, reveal that the presumed triad of selenocysteine, glutamine and tryptophan residues represents a novel type of catalytic centre, whose integrity is essential for the full catalytic function of glutathione peroxidases.

Direct measurement by single photon counting of lipid hydroperoxides in human plasma and lipoproteins.

A single photon counting procedure for measuring lipid hydroperoxides in human plasma or LDL-VLDL, escaping from extraction and chromatography, is described. This appears to be a relevant procedure because the recovery of phospholipid hydroperoxides from plasma is a critical point which, in our hands, was limited and poorly reproducible. The sample is added to a reaction mixture containing luminol, hemin, and Triton X-100 in an alkaline buffer, the photon emission is recorded, and the data are processed using the monoexponential decay of the photon emission rate. The measurement is applied to (a) plasma passed through a "desalting" cartridge to eliminate the small water-soluble antioxidants which inhibit the chemiluminescent process or (b) apo-B-containing lipoproteins (LDL-VLDL) isolated by heparin-Sepharose affinity chromatography. The content of lipid hydroperoxides is calculated using an internal calibration with palmitoyllinoleoylphosphatidylcholine hydroperoxide. This procedure, based on a single photon counting technology, was adopted to produce reliable results using samples from which inhibitors of the photon emission process have not been completely eliminated. The specificity of the signal for lipid hydroperoxides was validated by its complete disappearance following incubation of the sample with glutathione and phospholipid-hydroperoxide glutathione peroxidase (EC 1.11.1.12), the sole enzyme specific for all classes of lipid hydroperoxides in lipoproteins. The interassay variability was < 10%. The results indicated that the concentration of lipid hydroperoxides in the plasma of 20 healthy subjects was 353 +/- 78 nM. In different subjects, LDL-VLDL accounted for 40-80% of the lipid hydroperoxides in plasma.

Measurement of lipid hydroperoxides in plasma lipoproteins by a new highly-sensitive 'single photon counting' luminometer.

The lipid hydroperoxide content of isolated, native human plasma lipoproteins, was measured, by the luminol-based chemiluminescent reaction, using a highly sensitive single photon counting instrument. The reaction was specific for lipid hydroperoxides since the signal completely disappeared after treatment with the selenoperoxidase specific for lipidic substrates. In this analytical procedure the whole kinetic of photon emission induced by lipid hydroperoxides and hemin in the presence of luminol is integrated, taking advantage of the mono-exponential fitting of the decay of photon emission. The addition of a detergent to the reaction mixture improved the precision of the measurements apparently by preventing oxidative chain reactions affecting the shape of the decay of photon emission. The sensitivity of the instrument allowed measurements on samples containing just a few picomoles of hydroperoxides, small enough to minimize the effect of antioxidants and quenchers possibly present in the sample (as in the case of lipoproteins). Thus, by using an internal calibration with a phospholipid hydroperoxide, the evaluation of the lipid hydroperoxide content in whole, native lipoproteins was possible without previous extraction and chromatographic separation. Data obtained from plasma lipoproteins isolated by different procedures suggest that lipid hydroperoxide content increases during ultracentrifugation.

Copper-induced lipid peroxidation in liposomes, micelles, and LDL: which is the role of vitamin E?

Liposomes, containing phospholipid hydroperoxides, are peroxidised in the presence of Cu++. Peroxidation starts after a period of resistance to oxidation, which is abolished by the shift of lipid organisation from bilayer to micellar dispersion. Independently from ongoing peroxidation, vitamin E in liposomes also reacts with Cu++, and it is consumed. The evidence that phospholipid hydroperoxides induce an acceleration of vitamin E consumption rate and that the consumption of vitamin E and phospholipid hydroperoxides are stoichiometric indicates that, in liposomes, the rate-limiting reaction is the interaction between radicals generated by copper from vitamin E and phospholipid hydroperoxides. In micelles, on the other hand, vitamin E is directly oxidised by copper at a much faster rate; thus, the concerted consumption of phospholipid hydroperoxides does not take place. Moreover, in micelles challenged with Cu++, vitamin E plays a pro-oxidant effect (M. Maiorino et al. FEBS Letts., 330(2):174-176; 1993). In LDL, incubation with Cu++ promotes vitamin E consumption at a fast rate, as in micelles, but not the concerted disappearance of lipid hydroperoxides, as in liposomes. However, the direct vitamin E oxidation by Cu++, observed in micelles and liposomes, does not lead to a pro-oxidant effect in LDL. The kinetics of peroxidation, indeed, is identical in native and vitamin E-depleted LDL. These results argue against an involvement of vitamin E, both as antioxidant or pro-oxidant in LDL challenged with Cu++, and suggest that other factors, besides antioxidant content, must be relevant in determining LDL oxidative resistance.

Purification and characterization of phospholipid hydroperoxide glutathione peroxidase from rat testis mitochondrial membranes.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is highly expressed in rat testis, where it is under gonadotropin control. In this organ a relevant PHGPx activity is strongly linked to mitochondria of cells undergoing differentiation to spermatozoa. This prompted a study on the possible difference between the soluble and the mitochondrial enzyme and the nature of the binding. The mitochondrial PHGPx activity could be solubilized by detergents or by the combined action of mild detergent treatment and ionic strength, thus suggesting an electrostatic binding of the protein to the inner surfaces of the organelle. The same chromatographic purification procedures were applied to cytosolic and membrane bound PHGPx, without revealing any significant difference between the two forms. Moreover, the electrophoretic mobility, the reactivity to antibodies and the fragmentation patterns also suggested the identity of the two forms of testis PHGPx. Eventually, testis cytosolic and membrane bound PHGPx showed the same substrate specificity for both peroxidic and thiol substrates. On the other hand, a complex behaviour on hydrophobic interaction chromatography, compatible with multiple forms of the enzyme, and with a different tertiary structure of the major peaks was observed for soluble and mitochondrial PHGPx. Accordingly, two-dimensional electrophoresis followed by immunostaining with monoclonal antibodies, showed the presence of multiple isoforms with a different pattern between the soluble and the mitochondrial enzyme. These differences are not accounted for by glycosylation or a different degree of phosphorylation of tyrosines. In both enzymes, indeed, no glycosylation was detected and no more than 10% of PHGPx molecules were shown to contain a phosphotyrosine residue.

A novel antioxidant flavonoid (IdB 1031) affecting molecular mechanisms of cellular activation.

In searching for new drug candidates which could help bridge the gaps between free radical oxidations, pathophysiological responses, and pharmacological treatment, a series of flavonoids was screened. The most interesting compound emerging from this screening, the flavone 3'-hydroxyfarrerol (IdB 1031), is presented in this article. This compound is a good inhibitor of microsomal lipid peroxidation induced by either iron-adenosine 5'-diphosphate (ADP) or carbon tetrachloride. The elevated rate constant for the interaction with peroxyl radicals, analysed by the kinetics of inhibition of crocin bleaching in the presence of a diazo initiator, gives an account for the observed antioxidant capacity. When tested on human neutrophils activated by fMLP, IdB 1031 inhibits (ID50:20 microM) respiratory burst. This effect, which is possibly linked to the observed inhibition of protein-kinase C (ID50:50 microM), seems rather specific since IdB 1031 does not inhibit tyr-kinases and casein-kinase-2, while Quercetin and other flavonoids inhibit unspecifically all these enzymes. These effects, as a whole, depict this compound as a drug candidate for diseases in which peroxidative damage is associated with the induction of inflammatory responses and specifically with activation of a respiratory burst of leucocytes.

Phospholipid-hydroperoxide glutathione peroxidase. Genomic DNA, cDNA, and deduced amino acid sequence.

The complete amino acid sequence of the selenoprotein phospholipid-hydroperoxide glutathione peroxidase (PHGPX) from pig heart has been deduced from the corresponding genomic DNA, the cDNA covering the coding region, and by sequencing the N terminus of the protein. The maximum length of the peptide chain derived from the cDNA amounts to 170 amino acid residues. By protein sequencing the N-terminal residues methionine and cysteine of the deduced sequence were found to be cleaved. The molecular mass of 19,671 Da obtained by laser desorption mass spectroscopy, however, significantly exceeds the mean molecular mass of 19,257.09 calculated for the sequence 3-170 of PHGPX, thus indicating posttranscriptional modification. In contrast to glutathione peroxidase (GPX) the coding area of the PHGPX gene is composed of seven exons. Only the amino acid sequences encoded by the third and fifth exon are highly homologous to GPX sequences. The amino acid residues selenocysteine, tryptophan, and glutamine forming the catalytic site in bovine GPX are conserved in homologous positions of PHGPX, whereas the arginine residues presumed to bind GSH in GPX are not. Gaps in the PHGPX sequence correspond to subunit interaction sites of the tetrameric GPX. The data suggest an identical catalytic mechanism of the selenoperoxidases, a less stringent substrate specificity of PHGPX, and explain the monomeric nature of PHGPX. As in other selenoproteins, the selenocysteine residue of PHGPX is encoded by UGA. The 3'-untranslated region (UTR) of the PHGPX shows a limited consensus with that of GPX and 5'-deiodinase, where it was shown to be responsible for the decoding of UGA as selenocysteine. The 3'-UTR of PHGPX can form a stem/loop as in other mammalian selenoprotein genes. The 5'-UTR and the first intron of the PHGPX gene contain a variety of putative regulatory elements indicating hormonal control.

Phospholipid hydroperoxide glutathione peroxidase in the normal human kidney: a possible role in protecting cell membranes.

Reactive oxygen species have been implicated in the pathogenesis of tissue injury. It is generally accepted that selenium-glutathione peroxidases form an integrated system defending the living organism against oxidative damage. Phospholipid hydroperoxide glutathione peroxidase (PHGPX) is thought to play a prominent role in preventing lipid peroxidation. Indeed, the function of PHGPX is to reduce the lipophilic substrates in membranes. In the present study, we evaluated the expression of PHGPX in normal human kidney by immunohistochemistry. The enzyme in glomeruli is mainly expressed in podocytes and parietal epithelial cells. In addition, PHGPX antigen was detected in tubule epithelial cells. Therefore, these results suggest that renal epithelial cells possess an important antioxidizing activity related to the presence of PHGPX.

Prooxidant role of vitamin E in copper induced lipid peroxidation.

When exposed to Cu2+, alpha-tocopherol, in detergent dispersion, is rapidly oxidised. Moreover, if phospholipids and traces of their hydroperoxide derivatives are included in these dispersions, Cu2+ initiates lipid peroxidation, the rate of which is dramatically stimulated by alpha-tocopherol. The observation that the rate of alpha-tocopherol consumption is identical in the absence and in the presence of lipids undergoing peroxidation, apparently rules out any antioxidant effect. These results are consistent with a prooxidant effect of vitamin E, mediated by its capability to reduce Cu2+ to Cu+ which, in turn, produces, from lipid hydroperoxides, the highly reactive alkoxyl radicals. Present data highlight the risk of misleading results in interpreting the significance of lags in peroxidation of LDL challenged with Cu2+.