Junichi Fujii

Advances in our understanding of peroxiredoxin, a multifunctional, mammalian redox protein

Abstract Organisms living under aerobic conditions have developed various anti-oxidative mechanisms to protect them from damage by reactive oxygen species (ROS). A novel family of anti-oxidative proteins, designated as peroxiredoxin (Prx), has been identified in the past two decades and currently comprises six members in mammals. They share a common reactive Cys residue in the N-terminal region, and are capable of serving as a peroxidase and involve thioredoxin and/or glutathione as the electron donor. Prx1 to Prx4 have an additional Cys residue in the conserved C-terminal region, and are cross members as judged by the amino acid sequence similarity. Prx5 also contains an additional Cys in its C-terminal region which is less conserved. On the other hand, Prx6 has only one unique Cys. These Prx family members are distributed in the cytosol, mitochondria, peroxisome and plasma, all of which are potential sites of ROS production. In addition to their role as a peroxidase, however, a body of evidence has accumulated to suggest that individual members also serve divergent functions which are associated with various biological processes such as the detoxification of oxidants, cell proliferation, differentiation and gene expression. It would be expected that these functions might not necessarily depend on peroxidase activity and, therefore, it seems likely that the divergence is due to unique molecular characteristics intrinsic to each member. A comparative study of the divergence would lead to a better understanding of the biological significance of the Prx family.

Increased protein glycation in cerebrospinal fluid of Alzheimer’s disease

Accumulation of advanced glycation end products occurs in the brain with ageing and was proposed to be involved in pathogenesis of Alzheimers disease. We studied changes in the level of an early glycation product, an Amadori product, in cerebrospinal fluid (CSF) in ageing and in late-onset Alzheimers disease. The work was carried out on 99 consecutive patients. The concentration of Amadori product in CSF correlated with CSF glucose concentration but was not changed with age (n = 70). In contrast, level of CSF Amadori product was 1.7-fold higher in Alzheimers disease patients (n = 29) as compared with non-demented age-matched control group (n = 20; P < 0.0005), although CSF glucose concentration was similar in both groups (4.1 +/- 1.3 vs. 3.8 +/- 0.6 mmol/liter, resp.). An increased accumulation of Amadori products was found in all major proteins of CSF of Alzheimers disease including albumin, apolipoprotein E and transthyretin. We propose that the increased early glycation of CSF proteins in the Alzheimers patients may stimulate the formation and the consequent deposition of advanced glycation end products as well as oxidative stress in the brain.

Alteration of Glutathione Reductase Expression in the Female Reproductive Organs During the Estrous Cycle

Abstract The enzyme glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) in an NADPH-dependent manner. A specific antibody raised against recombinant rat GR was used to localize the protein in the female reproductive organs during the estrous cycle in the rat. In the ovary, the strongest reactivity to the antibody was observed in oocytes, followed by granulosa cells, corpus luteum, and interstitial cells. A strongly positive reaction was also observed mainly in the oviduct epithelia, uterine epithelia, and endometrial gland in the reproductive tract. Oviducts contained the highest GR activity. The GR activity of uterus during metestrus was about twice as high as that for other stages of the cycle. The levels of GR proteins in the tissues roughly matched the activities. The expression of the GR mRNA was highest during metestrus. Because GSH is known to increase gamete viability and the efficiency of fertility, GR, which is expressed in these tissues, is predicted to play a pivotal role in the reproduction process as a source of GSH.

Glycation proceeds faster in mutated Cu, Zn-superoxide dismutases related to familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. It has been shown that 15–20% of patients with familial ALS (FALS) have defects in the Sod1 gene that encodes Cu, Zn‐superoxide dismutase (SOD). To elucidate the pathological role of mutated Cu, Zn‐SODs in FALS, the susceptibility of mutants to glycation was examined. Mutated Cu, Zn‐SODs (G37R, G93A, and I113T) related to FALS and wild type were produced in a baculovirus/insect cell expression system. Glycated and nonglycated proteins were separated on a boronate column, and the nonglycated fraction was then incubated with glucose. The mutated Cu, Zn‐SODs were found to be highly susceptible to glycation compared with the wild‐type enzyme as estimated by Western blot analysis using an anti‐hexitol lysine antibody. The mutated Cu, Zn‐SOD incubated with glucose generated higher levels of hydrogen peroxide than the wild‐type enzyme. Mutated Cu, Zn‐SODs were also shown to be highly susceptible to fructation, and the fructated mutant also produced higher levels of hydrogen peroxide than the wild type. These results suggest that high susceptibility of mutated Cu, Zn‐SODs to glycation could be the origin of the oxidative stress associated with neuronal dysfunction in FALS.

Differential Expression of Glutathione Reductase and Cytosolic Glutathione Peroxidase, GPX1, in Developing Rat Lungs and Kidneys

A mutant rat GPX1 (a cytosolic predominant form), in which the selenocysteine residue in the catalytic center was replaced by cysteine, was prepared and an antibody against the mutant enzyme was raised. The resultant antibody specifically reacted with rat GPX1 and was, together with the Glutathione reductase (GR) antibody, used in a Western blot analysis and immunohistochemistry experiments. To elucidate the physiological coupling of these enzymes under oxidative stress which accompanies the birth, developmental changes of the protein levels and enzymatic activities of GR and GPX1 were examined for lungs and kidneys from prenatal fetus to adult rats. The expression of GR was already evident at the prenatal stage and remained high in lungs at all stages. However, GR activity in kidneys gradually increased after birth reaching maximal levels at adulthood. An immunohistochemical study showed that GR was strongly bound to the bronchial epithelia in lungs and the epithelial cells of renal tubes. GPX1 was expressed in the renal tube epithelial cells and its level gradually increased after birth in a manner similar to that of GR. The expression of GPX1 in the lungs was, on the other hand, variable and occurred in some alveolar cells and bronchial epithelia only at restricted periods. It preferentially localized in nuclei at a late stage of development. Thus, the expression of the two functionally coupled enzymes via GSH did not appear to coordinate with development, tissue localization or under oxidative stress. Since many gene products show GSH-dependent preoxidase activity, other peroxidase(s) may be induced to compensate for the low GPX1 levels at stages with high GR expression.

Impaired expression of peroxiredoxin 4 in damaged testes by artificial cryptorchidism

Abstract Peroxiredoxins (Prx) are a recently identified family of proteins that have been shown to exhibit peroxidase activity, as well as other divergent functions. Of the six known members of the family, Prx4 is present as a secretable form in most tissues and as a membrane-bound form only in testes. We recently proposed that the unprocessed form may be involved in acrosome formation during spermiogenesis. In the present study, it was found that levels of the unprocessed Prx4 decreased during cryptorchidism and this decrease corresponded to the degree of the defect in spermiogenesis. In contrast, the levels of the secretable Prx4 remained virtually the same during cryptorchidism. These results were consistent with selective expression of the unprocessed form after the spermatid stage, suggesting the existence of a relationship with acrosome formation.

Transient Elevation of Glutathione Peroxidase 1 Around the Time of Eyelid Opening in the Neonatal Rat

Glutathione peroxidase (GPX) reduces peroxides using reduced glutathione as the electron donor. Glutathione-dependent peroxidase activity in the soluble fraction of whole rat eye extracts (n = 3 or 4 at each stage) was the highest in the pre-natal stage (31.0 +/- 1.9 mU/mg protein) and gradually declined thereafter. The lowest value was 15.3 +/- 2.3 mU/mg protein at day 9. When the protein levels of the major selenium-containing glutathione peroxidase, GPX1, and the recently identified non-selenium-containing glutathione peroxidase, peroxiredoxin 6, were evaluated by immunoblotting using specific antibodies, they gradually declined after birth. An immunohistochemical analysis was carried out to identify the cells that express GPX1. Although the presence of GPX1 was evident only in restricted tissues, such as the corneal and lens epithelia in the adult, its levels were transiently augmented in ganglion cells, the layer of rods and cones, and pigment cells in the retina from 6 to 12 days after birth and then declined afterward. At the adult stage, the expression of GPX1 was negligible in these cells. Thus GPX1 appears to play a major role at this neonatal stage, corresponding to the period for eyelid opening. The decline in GPX1 levels after birth suggests that the detoxification of peroxides is important at this particular stage or that other, as yet unidentified peroxide-detoxifying enzymes are induced during this period.

Colocalization of polyol-metabolizing enzymes and immunological detection of fructated proteins in the female reproductive system of the rat.

The expression of aldose reductase (AR) and sorbitol dehydrogenase (SDH), which, in concert, catalyze the conversion of glucose to fructose via sorbitol, in the rat ovary, oviduct, and uterus, was investigated by immunohistochemical and biochemical analyses. The activities and protein levels of AR and SDH were higher in the ovary than in the oviduct and uterus. A strong immunoreactivity to the anti-AR antibody was observed in granulosa cells and epithelia of the oviduct, endometrium, and endometrial glands, and virtually the same tissues were strongly stained with the anti-SDH antibody. The application of an anti-fructated lysine antibody, which detects an adduct of fructose with the ε-amino group of lysine in proteins, in this study detected marked staining mainly in the egg and luminal surface of the oviductal epithelia. Collectively, these data indicate that fructose is produced by coordinately expressed AR and SDH in the egg and epithelia of the oviduct and suggest that the resulting sorbitol and fructose can be used as energy sources for spermatozoa motility during the fertilization process. The abundance of AR compared with SDH suggests that it also plays an additional role in the reproductive system, which might include a source of reducing power and protection against toxic carbonyl compounds.