Regina Brigelius-Flohé

Phospholipid Hydroperoxide Glutathione Peroxidase is a Seleno-Enzyme Distinct from the Classical Glutathione Peroxidase as Evident from Cdna and Amino Acid Sequencing

The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.

Selenoproteins of the glutathione system

The protein family of glutathione peroxidases (GPx) is found throughout the entire life kingdoms. Five distinct molecular clades characterized by an active site selenocysteine residue may coexist in vertebrates. All selenocysteine-containing GPx types reduce hydroperoxides with rate constants k’1 near 107 M−1 sec−1, while the cysteine homologs are poor peroxidases. The scope of accepted hydroperoxides increases from the cytosolic and gastrointestinal type to the extracellular type and phospholipid hydroperoxide GPx, while the specificity for GSH declines in this order. Compelling evidence defines cGPx as a device to detoxify H2O2 and soluble hydroperoxides. Being dispensable for survival, cGPx is nevertheless essential to maintain hydroperoxide homeostasis, as demonstrated by mimicking the development of Keshan disease in cGPx(−/−) mice. Being unable to substitute for cGPx in challenged cGPx(−/−) mice, the functions of other GPxs have to be sought in local regulation of peroxide-dependent processes, e. g., silencing leukotriene biosynthesis, dampening cytokine-dependent NFκB activation, regulating apoptosis and sperm differentiation by PHGPx. The functional divergence within the GPx family is further underscored by different mechanisms of transcriptional control.

Regulation of Glutathione Peroxidases

The glutathione peroxidases (GPx) belong to a superfamily of phylogenetically related proteins of diverse functions.1 The members of the superfamily containing a selenocysteine residue in their catalytic centers are highly efficient peroxidases reacting with a variety of hydroperoxides at rate constants of greater than 106 M-1 s-1.1 A cysteine residue in homologous position is catalytically less effective by about three orders of magnitude.2,3 Such GPx-like proteins, although still potential redox catalysts, can not be rated as peroxidases, if this term is to designate enzymes that must remove peroxides rapidly from a biological environment.

Selenium and male reproduction.

Selenium deficiency has long been documented to result in impaired male fertility of rats, mice and boars. The prominent feature of selenium-deficient spermatozoa is a distorted architecture of the mid piece, where normally the mitochondria are embedded into a keratinous matrix called the mitochondrial capsule. This material, which contains most of the selenium of sperm, is composed of oxidatively cross-linked proteins, a major component being the selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx). PHGPx is abundantly synthesized in round spermatids under indirect control of testosterone. In late phase of spermatogenesis, the active soluble peroxidase is transformed into an enzymatically inactive structural protein by an oxidative process that is not understood in detail. Likely, it involves oligomerization of PHGPx itself, cross-linking of PHGPx with the sperm mitochondrion-associated cysteine-rich protein (SMCP) and other cysteine-rich proteins and selenadisulfide reshuffling with or without the aid of thioredoxin-glutathione reductase.

Selenoproteins of the Glutathione Peroxidase Family

The glutathione peroxidase (GPx) family is spread over the entire living kingdom. In humans, eight distinct molecular paralogs coexist, five of which (GPx1, 2, 3, 4, and 6) contain Sec as the active-site residue. The selenoperoxidases (SecGPx) prevail in vertebrates, while GPx homologs having the active-site Sec replaced by Cys (CysGPx) are found in terrestrial plants, yeasts, protozoa, and bacteria. The typical signature of GPxs is an active-site tetrad composed of Sec or Cys, Trp, Gln, and Asn. SecGPx efficiently reduces hydroperoxides with rate constants, k +1, beyond 107 M-1 s-1, while the CysGPxs rarely reach a k +1 near 106 M-1 s-1. The scope of accepted hydroperoxides appears to broaden from GPx1 and GPx2 to GPx3 and phospholipid hydroperoxide GPx (GPx-4), while the specificity for GSH declines in this order. Most of the non-mammalian CysGPxs use redoxin-type proteins as reducing substrate. The scope of biological functions of GPxs comprises detoxification of hydroperoxides, inhibition of apoptosis and inflammatory processes, modulation of signaling cascades, sensing of H2O2 for activation of transcription factors, and using ROOH for the synthesis of structural proteins.

High expression vectors for the production of recombinant single-chain urinary plasminogen activator from Escherichia coli

SummaryAn expression cassette containing a synonymous gene for human single-chain urokinase-type plasminogen activator (Rscu-PA) 5-flanked by a trp promoter and the Shine-Dalgarno sequence of the xyl A operon of Bacillus subtilis and terminated by the terminators trp A and Tn10 was constructed and inserted into a pBR322 derivative to yield pBF160. When compared to pUK54 trp 207-1 containing the natural scu-PA gene without the Shine-Dalgarno sequence and terminator, the expression efficiency of pBF160 in Escherichia coli strains was improved by one order of magnitude. Replacement of the trp by the tac promoter (pBF171) did not affect expression. Inserting the Shine-Dalgarno sequence and Tn10 terminator into pUK54 trp 207-1 (pWH1320) slightly increased the expression level, whereas elimination of the Shine-Dalgarno sequence and the terminators from pBF160 with almost complete conservation of the synonymous structural gene (pBF191) significantly reduced the expression. Variation of the distance between the Shine-Dalgarno sequence and the start codon between 8 and 10 bp (pBF163) proved irrelevant. In conclusion, poor expression of mammalian genes in E. coli may result from both improperly designed regulatory elements and structural features of the coding region and therefore de-novo synthesis of the gene may be required to obtain satisfactory expression.

Basics and News on Glutathione Peroxidases

The catalytic mechanism of glutathione peroxidases and its variations in the subfamilies are reviewed and biological roles of the individual enzymes are compiled. The oxidative part of the catalytic cycle involves a water-mediated charge separation in the reaction center leading to dissociation of the selenocysteine (or cysteine) residue and binding of the delocalized proton in a highly energized position. In this environment, a suitably bound H2O2 is cleaved without any energy barrier in a concerted reaction yielding water and a selenenic (sulfenic) acid. Depending on family subtype and physiological conditions, the unstable oxidized enzymes form intramolecular disulfide or selenenylamide bonds. The reductive part of the cycle involves the reaction of selenenic (sulfenic) acid with a thiol and (selena) disulfide exchange. Trivial in principle, the reduction steps are most variable within the family, which explains its diversified specificities ranging from GSH to thioredoxin, disulfide isomerases, and particular SH groups of other proteins. The versatility in substrate and co-substrate use predestines these proteins for redox regulation, either as competitors for hydroperoxide utilization by other regulatory proteins or as sensor(s)/transducer(s) in hydroperoxide-initiated signaling cascades.