Interconnection between the structure and protective action of normal and pathological ceruloplasmin preparations in copper-induced erythrocyte lysis]

It was found that the differences in the protective effects of ceruloplasmin (CP) isolated from the blood of healthy donors and of the ceruloplasmin-like protein (pat-CP) isolated from the blood of patients with hepatovertebral dystrophy (HCD) during Ca(2+)-induced lysis of erythrocytes (RBC) result from significant changes in the carbohydrate fragment of pat-CP, the bulk of which (65%) is devoid of mannose and acetylglucosamine residues. According to the data from lentil-lectin Sepharose chromatography, only 4% of pat-CP molecules contain the [formula; see text] fragment necessary for the binding to ER receptors. The curves reflecting the Cu2+ accumulation in healthy donor ER and in pat-CP during the Cu(2+)-induced lysis were found to differ significantly. The ability of pat-CP to prevent the accumulation of Cu2+ in ER and pat-ER was markedly decreased compared with CP. Besides, CP prevented the diminution of reduced glutathione (GSH) in ER in a greater degree than pat-CP, whereas pat-ER, in contrast with CP, had no effect on the GSH concentration in pat-ER. It is suggested that the reactions occurring in the cell during Cu(2+)-induced lysis of ER and pat-ER are different.     

The protective effect of different forms of human ceruloplasmin in copper-induced lysis of red blood cells

The comparison of protective effects of native ceruloplasmin (CP) and of preparation CP1 containing carbohydrate fragment GlcNAc(beta(1,4]GlcNAc which specifically binds on RBC (alpha(1,6)Fuc receptors showed that CP1 exhibits much more powerful protective effect on RBC in copper-induced lysis. It was found, however, that CP2 (native CP devoided of CP1) protected RBC as well as CP despite its inability of binding to RBC membrane. CP and CP1 in a similar way decrease copper concentration in RBC. It was shown that copper accumulation and GSH decrease in RBC are two independent and concurrent processes; the copper and GSH concentrations are not the factors determining RBC resistance to hemolysis. CP inhibits the reaction of superoxide radicals generation as a result of Cu interaction with -SH groups of RBC membrane; the effect is more pronounced than the effect of catalase or superoxide dismutase. CP and CP1 preparations equally inhibit this reaction. Apparently CP reception on RBC leads not only to membrane protection from superoxide and hydroxyl radicals but represents a more complex process.     

Interrelation between structure and protective action of normal and pathological ceruloplasmins during copper-induced lysis of red blood cells

The origin of the difference between the protective action of ceruloplasmin (CP) from healthy donors blood and of ceruloplasmin-like protein (p-CP) from blood of patients with Wilson disease which they exert during copper-induced lysis of red blood cells (RBC) was elucidated. The difference is due to a significant change in the carbohydrate moiety of p-CP the major proportion of which (65%) does not contain mannose and acetylglucosamine residues. The data of chromatography on lentil lectin reveal that only 4% of p-CP molecules contain the fragment [table: see text] required for binding to RBC receptors. It was shown that the time-courses of copper accumulation in RBC of normal donors and in RBC of patients with Wilson disease (p-RBC) during copper-induced lysis differ markedly from each other. The p-CP is able to prevent copper accumulation in RBC and p-RBC to a significantly less degree than CP. It was also established that CP prevents the decrease of reduced glutathione (GSH) level in RBC to a greater extent than p-CP. In contrast to CP, the p-CP exerts no effect on the decrease in GSH concentration in p-RBC. These results may indicate that no interaction between Cu2+ and reduced glutathione takes place in p-RBC, in contrast to the situation occurring in normal RBC.     

The effect of copper ion on glutathione and hemolysis in rabbit erythrocytes

The rate of hemolysis and the decline in glutathione (GSH) in rabbit erythrocytes caused by copper (Cu) ions were determined. Prior investigations have proposed that the oxidative stress induced by Cu ion depleted the normal cell protective mechanisms. The decline in GSH has been proposed as a necessary prerequisite for hemolysis. We have observed that both GSH decline and hemolysis are Cu dependent, but are two concurrent and independent processes. We have confirmed that oxygen is a necessary reactant for hemolysis and responsible for a major portion of GSH decline. However, in the presence of Cu ion, a slow decline in GSH occurs even in a deaerated system.     

Protective effect rendered by human ceruloplasmin on erythrocytes in hepatocerebral dystrophy

In order to elucidate the protective effect of human ceruloplasmin (CP) on erythrocytes in patients with hepatocerebral dystrophy (HCD), the parameters reflecting the interaction of CP from the blood of healthy donors (n-CP) and of HCD patients (h-CP) with erythrocytes from healthy donors (n-ER) and from HCD patients (h-ER) were estimated. The protective effects of n-CP and h-CP on n-ER and h-ER during the Cu2+-induced lysis were compared. It was shown that the ability of h-CP to prevent the human ER breakdown upon Cu2+-induced lysis is much lower (approximately 3-fold) than that of n-CP. The differences in the protective effect of n-CP and h-CP are manifested in a greater degree during the n-ER lysis than during the h-ER lysis.     

Studies on receptor interaction of ceruloplasmin with human red blood cells

The interaction of CP with the red blood cell (RBC) receptor was shown to be a Ca2+ dependent process and be limited by CP binding on RBC membrane which is not followed by CP transport through the membrane into RBC. The nature of receptor interaction was determined. It was shown that receptors are formed by glycoproteins of PAS1 and PAS2 (glycoforin dimer and monomer, respectively) and terminal residues of sialic acid of these glycoproteins are important for CP reception. Receptor carbohydrate specificity was determined. Biantennary structure of CP molecule carbohydrate moiety which is bound to the receptor owing to 2 structural fragments: sialic acid terminal residues and the fragment including acetylglucosamine dimer and fucose, plays the main role in CP reception.     

Active fragments obtained by cyanogen bromide cleavage of ovomucoid.

Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule.     

Cys-His proteases are among the wired proteins of the cell

Integrated cell protein degradation can be paced by the transfer of reductive energy, as revealed by experimental agents of informative actions. The peptidolytic pair of Cys-His proteases can undergo oxidative reactions to inactive derivatives and inhibitory metal binding. Proton-dependent ionizations can modify ongoing activity. If the reaction rate of a Cys-His protease were found responsive to the ranges of metal/redox/proton factors regulated within the cell, then these factors might serve to link the peptidolytic reaction rate to cell controls. Here, cathepsin B (cat B) was found to be inhibited by Zn2+, Fe3+, and Cu2+ (1-50 microM) under excess GSH or DTT protease activators (6 mM). Under DTT or GSH (6 mM) the initial inhibitory action of Zn2+ is stable indefinitely; however, the inhibitory actions of Fe3+ and Cu2+ are reversed over approximately 1h. The 12-14 min half time of reversal of initial protease inhibition is correlated with the measured reduction of Fe3+ to Fe2+ by DTT or GSH (pH 5.5 or 6.5). Endogenous Fe2+ concentrations (100 microM) inhibit cat B only marginally. However, the inhibitory threshold of several microM Fe3+ is only a few percent oxidation of the endogenous pool. Without metals cat B reaction is reportedly proportional to GSH concentration, and is inhibited by increasing GSSG/GSH redox ratio. Following activation with GSH, cat B can be influenced by Fe3+/Fe2+, Cu2+/Cu+, and GSSG/GSH ratios and concentrations. Results are interpreted in relation to properties of the thiolate-imidazolium pair as illustrated by Dock modeling of their shared Fe3+ binding. It is proposed that the interaction of Cys-His with 1 electron transition between Fe2+ and Fe3+ serves as a sensor, signal integrator and switch wiring cat B reaction rate to the transfer of reductive energy in the presence of excess GSH. Speciated metals might also serve among electron acceptors transferring from reduced protease to oxygen. Results provide a model for pharmacologic redox switching of protease functions with metal-interactive drugs, and other nano-technology engineering.     

GSH inhibits trypsinization of the C-terminal half of human MRP1

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an approximately 35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and co-expressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L(0) region of MRP1. Thus the L(0) region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates.     

Human oestrogen receptors: differential expression of ER alpha and beta and the identification of ER beta variants

Two structurally related subtypes of oestrogen receptor (ER), known as alpha (ER alpha, NR3A1) and beta (ER beta, NR3A2) have been identified. ER beta mRNA and protein have been detected in a wide range of tissues including the vasculature, bone, and gonads in both males and females, as well as in cancers of the breast and prostate. In many tissues the pattern of expression of ER beta is distinct from that of ER alpha. A number of variant isoforms of the wild type beta receptor (ER beta 1), have been identified. In the human these include: (1). use of alternative start sites within the mRNA leading to translation of either a long (530 amino acids, hER beta 1L) or a truncated form (487aa hER beta 1s); (2). deletion of exons by alternative splicing; (3). formation of several isoforms (ER beta 2-beta 5) due to alternative splicing of exons encoding the carboxy terminus (F domain). We have raised monoclonal antibodies specific for hER beta1 as well as to three of the C terminal isoforms (beta2, beta 4 and beta 5). Using these antibodies we have found that ER beta 2, beta 4 and beta 5 proteins are expressed in nuclei of human tissues including the ovary, placenta, testis and vas deferens.In conclusion, in addition to the differential expression of full length ER alpha and ER beta a number of ER variant isoforms have been identified. The impact of the expression of these isoforms on cell responsiveness to oestrogens may add additional complexity to the ways in which oestrogenic ligands influence cell function.     

Effects of copper ions on the free radical-scavenging properties of reduced gluthathione: implications of a complex formation

The interaction between glutathione (GSH) and copper ions was investigated in vitro to determine whether such interaction could affect the free-radical scavenging properties of the tripeptide. To this end, the bleaching (decrease in OD734 nm) of a coloured solution containing the stable free-radical cation ABTS+, (which results from the addition of thiols to such a solution) was employed as an in vitro indication of the ability of the tripeptide to scavenge free radicals. While GSH bleached concentration-dependently (1.0-7.5 gM) the ABTS+-containing solution, its prior incubation (5 microM) in the presence of Cu+1 or Cu+2 ions (1-7.5 M) led to a metal concentration-dependent decrease of the bleaching capacity. At a ratio equal to one (5 microM each), the bleaching capacity of the copper plus GSH mixture was 50% of that seen for GSH alone. Further additions of copper (reaching ratios up to 2) did not result in greater decreases in the GSH-bleaching capacity. Noteworthy at the ratio of onewas that the copper plus GSH solutions maintained their bleaching capacity despite the lack of any DTNB-reactivity, i. e., the complete absence of thiols in the mixture. Mixtures of increasing concentrations of a fixed ratio (equal to 2) of copper plus GSH, which were found not to exhibit any DTNB-reactivity, showed a linear and concentration-dependent increase in bleaching capacity. The bleaching capacity remained unaltered when TRIEN, EDTA or histidine were added to pre-incubated (1:1) mixtures of copper plus GSH. However, the incubation of copper with TRIEN or EDTA (but not histidine) prior to GSH addition, totally prevented the loss of the original GSH-bleaching capacity. The present data supports the formation of a copper-glutathione complex which is stable to the presence of some copper-chelators, lacks all thiol reactivity, but fully conserves the free-radical scavenging properties of GSH.     

The effects of nitric oxide-oxidase and putative glutathione-peroxidase activities of ceruloplasmin on the viability of cardiomyocytes exposed to hydrogen peroxide

Ceruloplasmin (CP), a ferroxidase (EC 1.16.3.1) and a scavenger of reactive oxygen species, is an important extracellular antioxidant. Bovine CP indeed protects the isolated heart under ischemia-reperfusion conditions. Human CP has been shown to also exhibit, in vitro, glutathione (GSH)-peroxidase and nitric oxide (NO)-oxidase/S-nitrosating activities. This work tested, using bovine CP, the hypothesis that both activities could provide cytoprotection during oxidative stress induced by hydrogen peroxide (H(2)O(2)), the former activity by consuming H(2)O(2) and the latter by shielding thiols from irreversible oxidation. In acellular assays, bovine CP stimulated the generation of the nitrosating NO(+) species from the NO donors propylaminepropylamine-NONOate (PAPA/NO), S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione. This NO-oxidase activity S-nitrosated GSH as well as CP itself and was not affected by H(2)O(2). In contrast to human CP, bovine CP consumed H(2)O(2) in an additive rather than synergistic manner in the presence of GSH. A nonenzymatic scavenging of H(2)O(2) could have masked the GSH-peroxidase activity. Cytoprotection was evaluated using neonatal rat cardiomyocytes. CP and PAPA/NO were not protective against the H(2)O(2)-induced loss of viability. In contrast, GSH provided a slight protection that increased more than additively in the presence of CP. This increase was canceled by PAPA/NO. CP's putative GSH-peroxidase activity can thus provide cytoprotection but is possibly affected by the S-nitrosation of a catalytically important cysteine residue.     

N-Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface.

The primary structures of N-terminal 19-mer peptides, released by limited trypsin treatment of coat protein (CP) subunits in intact virions of three potato virus X (PVX) isolates, were analyzed. Two wild-type PVX strains, Russian (Ru) and British (UK3), were used and also the ST mutant of UK3 in which all 12 serine and threonine residues in the CP N-terminal segment were replaced by glycine or alanine. With the help of direct carbohydrate analysis and MS, it was found that the acetylated N-terminal peptides of both wild-type strains are glycosylated by a single monosaccharide residue (galactose or fucose) at NAcSer in the first position of the CP sequence, whereas the acetylated N-terminal segment of the ST mutant CP is unglycosylated. Fourier transform infrared spectra in the 1000-4000 cm(-1) region were measured for films of the intact and in situ trypsin-degraded PVX preparations at low and high humidity. These spectra revealed the presence of a broad-band in the region of valent vibrations of OH bonds (3100-3700 cm(-1)), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups. On calculating difference ('wet' minus 'dry') spectra, it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle. This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX. It is proposed that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation of a columnar-type shell from bound water molecules around the virions, which probably play a major role in maintaining the virion surface structure.     

Perturbation of nuclear–cytosolic shuttling of Rx1 compromises extreme resistance and translational arrest of potato virus X transcripts

Many plant intracellular immune receptors mount a hypersensitive response (HR) upon pathogen perception. The concomitant localized cell death is proposed to trap pathogens, such as viruses, inside infected cells, thereby preventing their spread. Notably, extreme resistance (ER) conferred by the potato immune receptor Rx1 to potato virus X (PVX) does not involve the death of infected cells. It is unknown what defines ER and how it differs from HR-based resistance. Interestingly, Rx1 can trigger an HR, but only upon artificial (over)expression of PVX or its avirulence coat protein (CP). Rx1 has a nucleocytoplasmic distribution and both pools are required for HR upon transient expression of a PVX-GFP amplicon. It is unknown whether mislocalized Rx1 variants can induce ER upon natural PVX infection. Here, we generated transgenic Nicotiana benthamiana producing nuclear- or cytosol-restricted Rx1 variants. We found that these variants can still mount an HR. However, nuclear- or cytosol-restricted Rx1 variants can no longer trigger ER or restricts viral infection. Interestingly, unlike the mislocalized Rx1 variants, wild-type Rx1 was found to compromise CP protein accumulation. We show that the lack of CP accumulation does not result from its degradation but is likely to be linked with translational arrest of its mRNA. Together, our findings suggest that translational arrest of viral genes is a major component of ER and, unlike the HR, is required for resistance to PVX.     

Two pathways for the degradation of the H2 subunit of the asialoglycoprotein receptor in the endoplasmic reticulum

An intermediate of 35 kD accumulates transiently during ER degradation of the H2 subunit of the asialoglycoprotein receptor; it is derived by an endoproteolytic cleavage in the exoplasmic domain near the transmembrane region. In the presence of cycloheximide all of the precursor H2 is converted to this intermediate, which is degraded only after cycloheximide is removed (Wikstrom, L., and H. F. Lodish. 1991. J. Cell Biol. 113:997-1007). Here we have generated mutants of H2 that do not form the 35-kD fragment, either in transfected cells or during in vitro translation reactions in the presence of pancreatic microsomes. In transfected cells the kinetics of ER degradation of these mutant proteins are indistinguishable from that of wild-type H2, indicating the existence of a second pathway of ER degradation which does not involve formation of the 35-kD fragment. Degradation of H2 in the ER by this alternative pathway is inhibited by TLCK or TPCK, but neither formation nor degradation of the 35-kD fragment is blocked by these reagents. As determined by NH2-terminal sequencing of the 35-kD fragment, formed either in transfected cells or during in vitro translation reactions in the presence of pancreatic microsomes, the putative cleavage sites are between small polar, uncharged amino acid residues. Substitution of the residues NH2- or COOH-terminal to the cleavage site by large hydrophobic or charged ones decreased the amount of 35-kD fragment formed and in some cases changed the putative cleavage site. Cleavage can also be affected by amino acid substitutions (e.g., to proline or glycine) which change protein conformation. Therefore, the endoprotease that generates the 35-kD fragment has specificity similar to that of signal peptidase. H2a and H2b are isoforms that differ only by a pentapeptide insertion in the exoplasmic juxtamembrane region of H2a. 100% of H2a is degraded in the ER, but up to 30% of H2b folds properly and matures to the cell surface. The sites of cleavage to form the 35-kD fragment are slightly different in H2a and H2b. Two mutant H2b proteins, with either a glycine or proline substitution at the position of insertion of the pentapeptide in H2a, have metabolic fates similar to that of H2a. These mutations are likely to change the protein conformation in this region. Thus the conformation of the juxtamembrane domain of the H2 protein is important in determining its metabolic fate within the ER.     

A novel noninvasive method for assessing glutathione-conjugate efflux systems in the brain

Brain efflux systems export such conjugated metabolites as glutathione (GSH) and glucuronate conjugates, generated by the detoxification process, from the brain and serve to protect the brain from harmful metabolites. The intracerebral injection of a radiolabeled conjugate is a useful technique to assess brain efflux systems; however, this technique is not applicable to humans. Hence, we devised a novel noninvasive approach for assessing GSH-conjugate efflux systems using positron emission tomography. Here, we investigated whether or not a designed proprobe can deliver its GSH conjugate into the brain. Radiolabeled 6-chloro-7-methylpurine (7m6CP) was designed as the proprobe, and [(14)C]7m6CP was prepared by the reaction of 6-chloropurine with [(14)C]CH(3)I as a model of [(11)C]CH(3)I. The radiochemical yield and purity of [(14)C]7m6CP were 10-20% and greater than 99%, respectively. High brain uptake (0.8% ID/g) at 1 min was observed, followed by gradual radioactivity clearance from the brain for 5-60 min after the injection of [(14)C]7m6CP into rats. Analysis of metabolites confirmed that the presence of [(14)C]7m6CP was hardly observed, and 80% of the radioactivity was identical to its GSH conjugate for 15-60 min. The brain radioactivity was single-exponentially decreased during the period of 15-60 min post-injection of [(14)C]7m6CP, and the first-order efflux rate constant of the conjugate, estimated from the slope, was 0.0253 min(-1). These results showed that (1) [(14)C]7m6CP readily entered the brain, (2) it efficiently and specifically transformed to the GSH conjugate within the brain, and (3) after [(14)C]7m6CP disappearance, the clearance of radioactivity represented the only efflux of GSH conjugate. We conclude that 7m6CP can deliver the GSH conjugate into the brain and would be useful for assessing GSH-conjugate efflux systems noninvasively.     

Age-associated decline in gamma-glutamylcysteine synthetase gene expression in rats

Although glutathione (GSH) concentration has been reported to diminish with age, the mechanism underlying such age-associated decline in the GSH content is not well understood. In this study, we compared the gene expression of both subunits of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, in young, adult, and old Fisher 344 rats. It was found that GCS activity was significantly decreased with increased age in liver, kidney, lung, and red blood cells (RBC). Parallel with the decreased enzyme activity, the protein and mRNA contents of both GCS subunits also changed inversely with age in liver, kidney, and lung, implying a decreased GCS gene expression during aging. Such a reduced GCS gene expression was accompanied by a decline in total GSH content without any change in cysteine concentration. Furthermore, the decreased GCS gene expression in old rats was not associated with a decline in the plasma insulin or cortisol level. This study showed, for the first time, that the expression of both GCS subunit genes was decreased in some organs of old rats, which would result in a reduced rate of GSH biosynthesis. Such decline in GSH synthetic capacity may underlie the observed decrease in GSH content during aging.     

Intervention of glutathione in pre-mutagenic catechol-mediated DNA damage in the presence of copper(II) ions

The catechol-mediated DNA damage in the presence of Cu(II) ions involves oxidation of guanine to 8-oxoguanine (8-oxoG) and DNA strand scission. It proceeds through the reactive oxygen species (ROS) generation. The mutagenicity of 8-oxoG lesions is due to its miscoding propensity reflected in GC→TA transversion taking place during the DNA repair process. To gain new insights into the nature of catechol-mediated DNA damage and its prevention, we have investigated the changes in DNA melting characteristics and 8-oxoG formation as the indicators of DNA damage in a model calf-thymus DNA system. A novel fluorescence method for DNA melting temperature determination, based on DAPI fluorescent-probe staining, has been proposed. The DNA melting-onset temperature has been found to be more sensitive to DNA damage than the standard melting temperature due to the increased width of the melting transition observed in oxidatively damaged DNA. We have found that the efficiency of Fenton cascade in generating DNA-damaging ROS is higher for catechol than for GSH, two strong antioxidants, mainly due to the much longer distance between ROS-generating radical group in GS to nucleobases than that of semiquinone radical group to nucleobases (2.1nm vs. 0.27nm), making the ROS transport from GSH an order of magnitude less likely to damage DNA because of short lifetime of HO radicals. The antioxidant and DNA-protecting behaviors of GSH have been elucidated. We have found that the redox potential of GSH/GSSG couple is lower than that of catechol/semiquinone couple. Hence, GSH keeps catechol in the reduced state, thereby shutting down the initial step of the catechol-mediated Fenton cascade. The catechol-induced DNA damage in the presence of Cu(II) ions has also been confirmed in studies of ON-OFF hairpin-oligonucleotide beacons.