Expression of ovotransferrin enhances tolerance of yeast cells toward oxidative stress.

Recently, we found that ovotransferrin (OTf) undergoes distinct self-cleavage in a redox-dependent process and exhibited in vitro superoxide dismutase (SOD)-like activity. In this study, we explore that the expression of OTf confers high tolerance to oxidative stress in yeast cells. The OTf gene was cloned into the vector pPICZB and was successfully expressed in methylotrophic yeast, Pichia pastoris KM71H. There was no growth difference between the non-transformed strain and recombinant strains harboring a mock vector (pPICZB) or the OTf gene carrying a vector (OTf-pPICZB). Intracellularly expressed OTf was found to undergo self-cleavage, producing a major fragment of 15 kDa, which corresponded to the disulfide kringle domain of the N-terminal lobe. The yeast OTf transformants exhibited strong tolerance to oxidative stress induced by either hydrogen peroxide (H2O2) or diethyl maleate (DEM). Further, OTf transformants showed higher intracellular reducing capacity and enhanced cytosolic reductase activity. This study is the first to describe the ability of OTf to confer in vivo antioxidative stress function within a complicated milieu of eukaryotic cells and provide novel insights for the potential of the OTf gene for molecular breeding of industrial yeast strains with high tolerance to oxidative stress.

Ovotransferrin expression and release by chicken cell lines infected with Marek's disease virus.

Mammals posses both serum transferrin and lactoferrin, whose functions are taken over in birds by ovotransferrin, displaying both iron transport and antibacterial activities. Ovotransferrin also exerts antiviral activity towards Marek's disease virus, an avian member of the herpes family of viruses. This virus infects lymphoid organs and induces the transcription of ovotransferrin in infected chicken embryo fibroblasts. However, it has not yet been established whether ovotransferrin gene transcription is linked to the release of the protein outside the cells or whether ovotransferrin expression and release also occurs in chicken lymphoblastoid cells in which the Marek's disease viral genome is integrated. Our results indicate that both serum and egg-white isoforms of ovotransferrin are expressed and released in the supernatants of chicken embryo fibroblast and lymphoblastoid cells in the absence of infection. Viral infection of chicken embryo fibroblasts caused a slight increase of ovotransferrin release, whereas viral reinfection of lymphoblastoid cells caused a remarkable ovotransferrin release in a virus concentration-dependent manner. These findings suggest that ovotransferrin release in vivo may play a crucial role in protecting the whole organism from viral infection spreading, and support the hypothesis that the antiviral activity of ovotransferrin is an important part of the innate immune response in birds, resembling the antiviral activity of lactoferrin in mammals.

Structural and functional characterization of ovotransferrin produced by Pichia pastoris.

Ovotransferrin is an egg white protein with complex disulfide and bilobal structures, which is derived from the same gene as chicken serum transferrin. We demonstrate here the structural and functional characteristics of bilobal ovotransferrin, produced at a high level using Pichia pastoris expression system. The recombinant protein was secreted into the medium, and the secretion signal peptide was processed correctly. The secretion level was almost 100 mg/l culture and the yield after purification by two-step anion exchange chromatography was 57 mg/l. The CD spectrum and fluorescence spectra indicate the correct folding of the recombinant protein. The analyses for the Fe3+ binding ability by urea-PAGE and visible absorption spectrum revealed that two Fe3+ sites exist in a recombinant ovotransferrin molecule as in the egg white protein. Endoglycosidases, such as endo-beta-N-acetylglucosaminidase H (Endo-H), peptide:N-glycosidase F (PNGaseF), and endo-beta-N-acetylglucosaminidase from Mucor hiemalis, showed differential activities for the native Fe3+-loaded, native Fe3+-free, and denatured forms of recombinant ovotransferrin; only the first enzyme displayed the cleavage ability for all the ovotransferrin forms. The results from the enzyme specificity and from the molecular weight difference for the intact and deglycosylated proteins were consistent with the view that recombinant ovotransferrin have one N-linked carbohydrate chain which mainly consists of two GlcNac and 10 mannoses.

Identification of choroidal ovotransferrin as a potential ocular growth regulator.

PURPOSE: In an effort to identify choroidal factors potentially involved in the regulation of ocular growth, proteins released into culture medium of organ-cultured choroids were compared between control eyes and eyes recovering from form deprivation myopia. METHODS: The choroids were obtained from the posterior poles of control and recovering chick eyes, and placed into organ culture containing ( 35)S-methionine/(35)S-cysteine. Culture medium was collected after 24 hours and proteins were separated and identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), fluorography, immunoprecipitation, western blot analysis and by amino acid sequencing. Choroidal proteins were tested for their effect on scleral proteoglycan synthesis by measuring (35)SO( 4) incorporation into scleral glycosaminoglycans (GAG) in vitro. Choroidal thickness and axial elongation were measured in control and recovering eyes using high frequency A-scan ultrasound. RESULTS: The synthesis of an 80 kD protein was greatly increased in the choroids of recovering eyes compared with those of control eyes. Amino acid sequencing and immunoprecipitation indicated that the newly synthesized 80 kD protein was ovotransferrin (transferrin, conalbumin). Ovotransferrin release into the culture medium by isolated recovering choroids was associated with a decrease in the rate of axial elongation in recovering eyes. When tested in vitro, ovotransferrin (500 ng/micro) inhibited scleral proteoglycan synthesis in the sclera by 62% in a dose-dependent manner. CONCLUSIONS: Chick choroids of recovering eyes synthesize and release ovotransferrin during the recovery from form deprivation myopia. Ovotransferrin significantly inhibited proteoglycan synthesis by the sclera, indicating that ovotransferrin may play a role in slowing the rate of vitreous chamber elongation and facilitating the recovery from induced myopia.

Effects of insulin-like growth factor-I, estrogen, glucocorticoid, and transferrin on the mRNA contents of ovalbumin and conalbumin in primary cultures of quail (Coturnix coturnix japonica) oviduct cells.

The effects of estrogen, dexamethasone, insulin-like growth factor-I (IGF-I), and transferrin on the messenger RNA (mRNA) contents of ovalbumin and conalbumin in primary cultures of quail oviduct cells were investigated. In the absence of one of the above hormones or factors, a decrease in ovalbumin mRNA was prominent. In particular, removal of IGF-I and transferrin caused a significant effect. Studies using a combination of estrogen, dexamethasone, IGF-I and transferrin indicated that IGF-I cooperates with estrogen or dexamethasone and transferrin works with dexamethasone. Specifically, IGF-I enhanced ovalbumin synthesis or increased cellular ovalbumin mRNA content depending on its concentration in the medium in the presence of estrogen. However, the effects of estrogen, dexamethasone, IGF-I, and transferrin were not similarly observed with conalbumin mRNA. These results show that ovalbumin synthesis is controlled by estrogen or glucocorticoid with IGF-I or transferrin and that cellular ovalbumin mRNA content is also regulated by these hormones or transferrin. In contrast, conalbumin synthesis and cellular content of conalbumin mRNA are not affected by these hormones under the conditions of the present study.

Cell proliferation, extracellular matrix mineralization, and ovotransferrin transient expression during in vitro differentiation of chick hypertrophic chondrocytes into osteoblast-like cells.

Differentiation of hypertrophic chondrocytes toward an osteoblast-like phenotype occurs in vitro when cells are transferred to anchorage-dependent culture conditions in the presence of ascorbic acid (Descalzi Cancedda, F., C. Gentili, P. Manduca, and R. Cancedda. 1992. J. Cell Biol. 117:427-435). This process is enhanced by retinoic acid addition to the culture medium. Here we compare the growth of hypertrophic chondrocytes undergoing this differentiation process to the growth of hypertrophic chondrocytes maintained in suspension culture as such. The proliferation rate is significantly higher in the adherent hypertrophic chondrocytes differentiating to osteoblast-like cells. In cultures supplemented with retinoic acid the proliferation rate is further increased. In both cases cells stop proliferating when mineralization of the extracellular matrix begins. We also report on the ultrastructural organization of the osteoblast-like cell cultures and we show virtual identity with cultures of osteoblasts grown from bone chips. Cells are embedded in a dense meshwork of type I collagen fibers and mineral is observed in the extracellular matrix associated with collagen fibrils. Differentiating hypertrophic chondrocytes secrete large amounts of an 82-kD glycoprotein. The protein has been purified from conditioned medium and identified as ovotransferrin. It is transiently expressed during the in vitro differentiation of hypertrophic chondrocytes into osteoblast-like cells. In cultured hypertrophic chondrocytes treated with 500 nM retinoic acid, ovotransferrin is maximally expressed 3 d after retinoic acid addition, when the cartilage-bone-specific collagen shift occurs, and decays between the 5th and the 10th day, when cells have fully acquired the osteoblast-like phenotype. Similar results were obtained when retinoic acid was added to the culture at the 50 nM "physiological" concentration. Cells expressing ovotransferrin also coexpress ovotransferrin receptors. This suggests an autocrine mechanism in the control of chondrocyte differentiation to osteoblast-like cells.

The effects of estrogen, insulin and dexamethasone on the synthesis and secretion of egg white proteins in primary cultured oviduct cells of laying Japanese quail (Coturnix coturnix japonica).

1. The effects of estrogen, insulin and dexamethasone on the synthesis of egg white proteins were investigated by employing primary cultured oviduct cells of laying Japanese quails. 2. It was demonstrated that oviduct cells require insulin and dexamethasone, besides estrogen, to synthesize and secrete egg white proteins maximally.

Steroid hormones induce cell proliferation and specific protein synthesis in primary chick oviduct cultures.

A rapid method to obtain large amounts of tubular gland cells from chick oviduct was developed. Combined collagenase and trypsin treatment allowed within 1.5 h complete dissociation of the magnum portion of the oviduct. By differential attachment of cells, fibroblasts were separated from tubular gland- and ciliated cells. Tubular gland cells attached within 18 h to plastic Petri dishes, had large secretory granules and grew very actively. The responsiveness of cells to hormones and/or antihormone was tested by measurement of cell proliferation and specific protein synthesis. After 7 days of culture in the presence of estradiol (50 nM) or progesterone (100 nM), cell growth was increased by approximately 50 and 35% respectively. Tamoxifen (100 nM) inhibited the estradiol induced growth stimulation, but had also negative effects of its own. The anti-progesterone (in mammals) RU 486, inactive per se, did not antagonize progesterone induced growth. Ovalbumin- and conalbumin synthesis after 4-5 days of cultures under different hormonal conditions was assessed after immunoprecipitation of newly synthesized [35S]methionine labelled proteins. In the presence of estradiol (50 and 100 nM), progesterone (50 nM), and both estradiol and progesterone together (50 nM of each), ovalbumin and conalbumin synthesis was increased, when compared to control cultures without hormones, or to oviduct fibroblasts. Hormonal stimulation of ovalbumin synthesis was also shown in cell supernatant and culture medium after gel electrophoresis.

Effects of progesterone and tamoxifen on glucocorticosteroid-induced egg-white protein synthesis in the chick oviduct.

A single injection of either natural (cortisol, corticosterone) or synthetic [dexamethasone (DEX), triamcinolone acetonide] glucocorticosteroids to estradiol-primed, withdrawn chicks, resulted in a dose-dependent increase in the relative rates of ovalbumin and conalbumin synthesis. The simultaneous injection of equal doses of DEX and progesterone resulted in an additive effect on the relative rate of ovalbumin synthesis at all doses tested (range: 0.05-15 mg/chick), even when the induction of ovalbumin synthesis was maximal at 6 h, for either hormone injected alone. Moreover, the simultaneous injection of DEX and progesterone yielded an additive effect on the relative rates of ovalbumin and conalbumin gene transcription. The nonsteroidal antiestrogen tamoxifen does not increase ovalbumin synthesis and only slightly increases conalbumin synthesis. The simultaneous injection of tamoxifen and DEX potentiated the effect of DEX on the relative rates of ovalbumin and conalbumin synthesis, and amplified the DEX-induced increase in the relative rates of ovalbumin and conalbumin gene transcription. These results were supported by morphological studies carried out after 4 days of stimulation, which showed an increased accumulation of secretory granules in the magnum cells of the oviducts of chickens treated by tamoxifen plus DEX, as compared to that observed in chickens injected with DEX alone. In conclusion, these results suggest that glucocorticosteroids likely act through a mechanism distinct from that of sex steroids, and may modulate the effects of the latter on egg-white protein synthesis.

Desensitization of the chick oviduct to estrogen: mediation at different levels of gene expression.

We have investigated the effects of changing the dosage or kind of estrogen administered to immature chicks on the synthesis of two egg white proteins, ovalbumin and conalbumin, and the accumulation of their mRNAs. The results suggest that the oviduct can become desensitized to estrogen. Ovalbumin accounted for 25% of oviduct protein synthesis in chicks treated with low dose of diethylstilbestrol (DES; 0.5 mg/day) for 14 days. Within several days after the dosage of estrogen had been increased 10-fold, ovalbumin synthesis fell to undetectable levels. The synthesis of conalbumin also became undetectable when the dosage of estrogen was increased. Throughout the above experiment both ovalbumin and conalbumin mRNA activity, as measured by cell-free translation, remained elevated. This implies that expression of the ovalbumin and conalbumin genes may be regulated at the level of translation. In a separate experiment, ovalbumin and conalbumin synthesis also decreased when chicks primed with DES pellets were given an increased dosage of estrogen. Ovalbumin synthesis fluctuated, but overall decreased from 25% of oviduct protein synthesis after priming with DES pellets to 16% of oviduct protein synthesis after 7-12 days of injections of estradiol benzoate (1 mg/day). At the same time, conalbumin synthesis decreased from 10% to 6% of oviduct protein synthesis. In contrast with the previous results, changes in ovalbumin and conalbumin mRNA activity paralleled changes in ovalbumin and conalbumin synthesis. Thus, not only can the oviduct become desensitized to estrogen, desensitization can be mediated at different levels of gene expression.

Translation and stability of ovalbumin messenger RNA injected into growing oocytes and fertilized ova of mice.

Growing mouse oocytes and fertilized ova were injected with chicken ovalbumin messenger RNA (mRNAov) and chicken conalbumin mRNA (mRNAcon) and cultured in vitro. Estimation of mRNAov and mRNAcon stability by hybridization of cDNAov and cDNAcon to extracted mRNA from injected oocytes and fertilized ova indicated a half-life of 147 and 366 h in the oocyte and 5 and 3 h in the fertilized ovum respectively. Stability of mRNAov was similar in the fertilized and unfertilized ovum. Oocytes injected with chicken ovalbumin mRNA were also labelled with [3H]leucine and ovalbumin synthesis was measured by immunoprecipitation. The amount of ovalbumin synthesized during the initial 7 h was less than during the period of 18-25 or 66-73 h postinjection. The greatest percentage of ovalbumin to total protein synthesis occurred between 66-73 h. Oocytes secreted 12% of the synthesized ovalbumin during each of the 7 h periods (0-7, 18-25 and 66-73 h) indicating a stable mechanism for secretion throughout the culture period. These studies demonstrate: a dramatic difference in stability of injected mRNA between the growing oocyte and the unfertilized or fertilized ovum, and a gradual increase in the translation of injected mRNA by the growing oocyte during in vitro culture.

Effects of serial hormone treatments on egg white protein synthesis. Further evidence of translational regulation.

The administration of either progesterone or estrogen to withdrawn chicks several hours after a first dose of estrogen affected ovalbumin synthesis differently than its mRNA levels [S. S. Seaver (1981) J. steroid Biochem. 14, 949-957]. This suggested that the hormones were regulating the translation of ovalbumin directly. In this paper we report that serial hormone treatments also affect the rates of synthesis of two other egg white proteins, conalbumin and ovomucoid. When progesterone was administered 4 h after estrogen, conalbumin synthesis decreased. When either progesterone or a second dose of estrogen was administered 12 h after the first dose of estrogen, conalbumin synthesis increased. Serial hormone treatments did not always affect all three proteins similarly. At later times, administering progesterone after estrogen decreased ovomucoid synthesis but did not affect conalbumin or ovalbumin synthesis. To determine if the serial hormone treatments affect egg white protein mRNA's in a similar way, changes in ovalbumin and conalbumin mRNA levels were quantified in a rabbit reticulocyte cell-free translation system and were compared to changes in ovalbumin and conalbumin synthesis as measured in chick oviduct tissue minces. When serial hormone treatments were 12 h apart, ovalbumin and conalbumin synthesis was 50-300% higher than that predicted by the changes in ovalbumin or conalbumin mRNA levels. This is further evidence that translation of both conalbumin mRNA and ovalbumin mRNA is directly regulated by steroid hormones.

Chick embryo spinal cord neurons synthesize a transferrin-like myotrophic protein.

Highly enriched cultures of chick embryo spinal cord neurons synthesize and secrete a protein which is immunoprecipitable by anti-ovotransferrin. Ovotransferrin, an iron-binding glycoprotein of Mr 80 000, is also shown to stimulate in vitro myogenesis of cultured chick embryo myotubes as measured by saturable dose-dependent increase in acetylcholine receptors. This effect is probably dependent on ovotransferrin's ability to donate iron to the cells. In many respects ovotransferrin is similar to 'sciatin', a myotrophic protein isolated from chicken sciatic nerves.

Glucocorticoid induction of egg white mRNAs in chick oviduct.

Glucocorticoids induce ovalbumin and conalbumin mRNA in oviducts from chicks withdrawn from prior estrogen treatment. The magnitude and the kinetics of the responses obtained, either in vivo or in vitro, are comparable to those obtained with estrogen or progesterone. With cultured oviducts, 1 nM dexamethasone is sufficient for half-maximal accumulation of nuclear receptors and partial induction of both mRNAs, while maximal levels of receptors and both mRNAs are achieved with 30 to 100 nM dexamethasone. Competition experiments show that dexamethasone acts by binding to a class of high affinity receptors distinct from the sex steroid receptors. Dexamethasone acts synergistically with estrogen, but not with progesterone.

Steroid receptors and effects of oestradiol and progesterone on chick oviduct proteins.

After a single injection of oestradiol benzoate (1.5 mg/kg) to oestrogen-withdrawn chickens, there was an increase in magnum wet weight, DNA polymerase alpha activity, adenosine-3',5'-monophosphate-dependent protein-kinase activity and estrogen-receptor concentration, as measured over 36 h. Besides these intracellular proteins, the secretory proteins ovalbumin and conalbumin were also augmented, and detailed time-course studies were performed. Early induction kinetics for ovalbumin and conalbumin synthesis, which differed for each protein, were independent of the dose of oestradiol benzoate injected if it exceeded 0.1 mg/kg. After 6 h for ovalbumin and 2 h for conalbumin, the induction curves diverged according to the dose of hormone administered and in correlation with the persistence of elevated nuclear oestrogen-receptor concentrations, a result confirmed with 11 beta-methoxy-17 alpha-ethynyloestradiol (R 2858), a powerful synthetic oestrogen. When oestradiol benzoate (1 mg/kg) and progesterone (3 mg/kg) were injected simultaneously, the rate of conalbumin sythesis, during the first 6-8 h, was lower than that observed in animals injected with oestradiol benzoate alone. However at later times conalbumin synthesis was greater in animals receiving both hormones than with oestradiol alone. In contrast, the rate of ovalbumin synthesis after the combined injection was higher than that induced by either hormone alone throughout the entire experimental period. In order to study further the synergistic and antagonistic activities of these two hormones, a single injection of progesterone (3 mg/kg) was administered 6, 12 or 18 h after 1.5 mg/kg oestradiol benzoate. Progesterone administration resulted in a reduction in cytoplasmic, nuclear and total oestrogen receptor concentration for at least 6 h when compared with the values in birds treated with oestrogen alone. DNA polymerase and protein kinase activities were also reduced during this period. Subsequently, all parameters increased, and by 18-24 h after progesterone treatment, reached values higher than those observed in animals receiving oestrogen alone.

DNA methylation: organ specific variations in the methylation pattern within and around ovalbumin and other chicken genes.

The restriction enzymes HhaI and HpaII, whose activity is inhibited by cytosine methylation within their recognition sites, have been utilised as probes to study methylation in the vicinity of the ovalbumin gene in DNA from various chicken tissues. This was complemented by a preliminary study of methylation in the regions of chicken ovotransferrin (conalbumin), ovomucoid and beta-globin genes. From our data we conclude that HaI or HpaII sites can be divided in 3 classes according to their pattern of methylation in different tissues. In the first class of sites (mV class) the extent of methylation varies in different tissues. The patterns obtained show that methylation at the sites located within and around the 3 genes which code for egg white proteins is in general lowest in oviduct of laying hen, where these genes are expressed. However some sites are not methylated (m- class) and others are 95 to 100% resistant (m+ class) to digestion by HhaI or HpaII in the DNAs of all the tissues which were tested. Our study has also revealed a remarkable number of allelic variants for the presence of HhaI or HpaII sites in the region of the ovalbumin gene.