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The administration of either progesterone or estrogen to withdrawn chicks several hours after a first dose of estrogen affected ovalbumin synthesis differently than its mRNA levels [S. S. Seaver (1981) J. steroid Biochem. 14,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.     

Heterogeneity of the 5' terminus of hen ovalbumin messenger ribonucleic acid.

The 5'-terminal sequence of hen ovalbumin mRNA was investigated using a novel labeling method. Ovalbumin mRNA was purified by hybridization to complementary DNA coupled to cellulose. The mRNA thus purified was shown to be 97.9% pure by hybridization with plasmid DNA containing sequences to the messengers coding for conalbumin and ovomucoid, the next two most abundant messengers of oviduct. After digestion with RNase T1 and alkaline phosphatase, 5'-terminal capped oligonucleotides were selected by binding to anti-m7G-Sepharose. These were then labeled using RNA ligase and [5'-32P]pCp, separated by two-dimensional gel electrophoresis, and sequenced by partial digestion with base-specific ribonucleases. A nested set of three capped oligonucleotides was identified. Their structures and relative abundances were m7GpppAUACAG, 3% m7GpppACAUACAG, 61+; and m7GpppGUACAUACAG, 36%.     

Immunoadsorption of specific chicken oviduct polysomes. Isolation of ovalbumin, ovomucoid, and lysozyme messenger RNA.

The messenger RNA coding for the egg white proteins ovalbumin, ovomucoid, and lysozyme were isolated by immunoadsorption of polysomes synthesizing these proteins. Monospecific antibodies against ovalbumin, ovomucoid, and lysozyme, raised in rabbits, were reacted with chicken oviduct polysomes. The antibody-polysome complexes were isolated by immunoadsorption onto sheep anti-rabbit antibodies coupled to an insoluble matrix. The specifically bound polysomes were eluted and the mRNA was obtained by poly(U)-Sepharose chromatography. The three specific RNAs were further purified by preparative gel electrophoresis. The purity of the mRNA preparations was demonstrated by analytical gel electrophoresis, the capability to direct the synthesis of specific protein products in a wheat germ cell-free system, and by hybridization to cDNA transcribed from mRNAoa and mRNAomu. Purified mRNAoa was shown to contain less than 0.1% mRNAomu and purified mRNAomu was about 99% pure with respect to mRNAoa. Purified mRNAly was contaminated with mRNAoa to 0.34% and with mRNAomu to 2.9%.     

Purification Process for the Preparation and Characterizations of Hen Egg White Ovalbumin,Lysozyme, Ovotransferrin,and Ovomucoid

Abstract

In this study, four major egg white proteins were purified by two step ion exchange chromatography followed by precipitation. Lysozyme and ovalbumin were separated with Q Sepharose Fast Flow anion exchange chromatography in the first step, resulting in two peaks of lysozyme and three peaks of ovalbumin with 87% and 70% purity by HPLC, respectively. Ovotransferrin was separated with CM-Toyopearl 650 M cation exchange chromatography in the second step, giving 80% purity. Ovomucoid was precipitated from the partial purified protein fraction from the first two steps, and concentrated in the final step to yield 90% purity. Protein recoveries were estimated to be 55, 21, 54, and 21% for lysozyme, ovotransferrin, ovalbumin, and ovomuciod, respectively. Lysozyme was identified from the purified peaks using zymogram refolding gel, whereas ovalbumin was identified by western blotting. Purified ovotransferrin and ovomucoid was identified by mass spectrometry. The results indicate that this purification process is adequate for preparation of lysozyme, ovalbumin, ovotransferrin, and ovomucoid, at least on a laboratory scale.     

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.     

Multiple mRNAs are generated from the chicken lysozyme gene

We have determined the DNA sequence of a 770 by Pst I fragment containing 450 nucleotides of the 5′ flanking region of the chicken lysozyme gene. S1-nuclease mapping was performed to localize the 5′ end of nuclear RNA containing lysozyme-specific sequences and of the mRNA. We present evidence that the 5′ noncoding region of the chicken lysozyme mRNA is heterogeneous in length. The 5′ termini of the different mRNAs map 29, 31 and 53 nucleotides upstream from their common initiation codon. The 5′ ends of lysozyme-specific nuclear RNAs map at positions similar to that of the mRNA. AT-rich regions and sequences similar to the E. coli RNA polymerase recognition sequence are found around 30 and 70 nucleotides upstream from each of these 5′ termini. The AT-rich regions differ, however, from the canonical Goldberg-Hogness box in that they do not contain the extremely conserved TATA sequence motif. Sequence comparison at the 5′ end of the lysozyme, conalbumin and ovalbumin genes reveals only one region of partial homology, 140 nucleotides upstream from the mRNA start sites.     

Raman spectroscopic study of the proteins of egg white

The Raman spectra of ovalbumin, ovomucoid, and conalbumin are reported. Spectral shifts in the conformationally sensitive amide I and amide III lines as a result of thermal denaturation indicate the formation of intermolecular β- sheets. A medium intensity line at 1260 cm^?1 in the spectra of ovomucoid and ribonuclease is demonstrated to contain a substantial contribution from tyrosine residues.     

The induction of ovalbumin and conalbumin mRNA by estrogen and progesterone in chick oviduct explant cultures

Estrogen pretreated chick oviduct tissue can be restimulated in vitro by physiological concentrations of estrogen and progesterone. The rates of synthesis of the major egg white proteins, ovalbumin and conalbumin, as well as the cellular levels of their respective mRNAs, increase after characteristic lag periods; this confirms previously reported results in vivo and demonstrates that both the lag phenomena and the mRNA induction are a function of the direct interaction of steroids with oviduct cells.The antagonistic action of progesterone on an estrogen-mediated induction of conalbumin mRNA also occurs in vitro, and the kinetics of this response are examined. Progesterone terminates the estradiol-induced accumulation of conalbumin mRNA within 30 min after addition to the medium; progesterone alone or in combination with estrogen, however, is capable of inducing conalbumin mRNA after a 4 hr lag period. The temporary nature of this antagonism and the fact that it does not occur with ovalbumin induction indicate the complexity of the oviduct's response to steroids.The role of protein synthesis in the induction of both ovalbumin and conalbumin was examined by including protein synthesis inhibitors in the culture medium. Puromycin, cycloheximide, emetine, pactamycin and high salt all block the induction of both ovalbumin and conalbumin mRNA when added together with either estrogen or progesterone. The effect of puromycin is reversible. After the drug is removed from the medium, the mRNA accumulation begins with the same characteristic lag period seen when no inhibitors are added. When given 2 hr after estrogen, puromycin stops the accumulation of conalbumin mRNA within 30 min, whereas cycloheximide and emetine allow the mRNA to accumulate for another 2 hr before causing complete inhibition. There is no effect of protein synthesis inhibitors on the number of estrogen receptors localized in the nucleus. The data suggest a direct link between protein synthesis and the steroid-induced accumulation of specific mRNAs in this system.     

Isolation of a nuclear ribonucleoprotein fraction from chick oviduct containing ovalbumin messenger RNA sequences

A method was developed for the isolation of a ribonucleoprotein fraction from chick oviduct nuclei that contains 70% of the pulse-labeled RNA. These fractions also contain about 1% of the nuclear DNA and have an average RNA to DNA ratio of about 4:1. The major nuclear RNP proteins of 32,000 M_r are present along with many additional proteins including histories. However, polysomal proteins and major oviduct cytoplasmic proteins are absent. Nuclei from fully stimulated chick oviduct contain about 3000 copies of ovalbumin messenger RNA sequences of which about 200 are in the RNP complexes: these complexes have sedimentation coefficients of 30 to 350 S and are resistant to disruption by EDTA.The level of ovalbumin mRNA sequences in these complexes reflects the overall rate of synthesis of this RNA. Withdrawal of estrogen leads to a parallel decline of nuclear estrogen receptors and ovalbumin mRNA sequences in the RNP complexes and a subsequent loss of cytoplasmic ovalbumin mRNA about three hours later. The 300-fold decrease in the level of ovalbumin mRNA sequences in these complexes and the eightfold decrease in stability of cytoplasmic ovalbumin mRNA account for the 2500-fold decrease in the level of cytoplasmic ovalbumin mRNA observed during withdrawal. Upon stimulation with estrogen, the kinetics of reappearance of ovalbumin mRNA sequences in the RNP complexes apparently accounts for the accumulation of cytoplasmic ovalbumin mRNA. Thus the nuclear RNP has some of the properties expected of nascent RNP complexes.The levels of ovalbumin and conalbumin mRNA sequences increase in the nuclear RNP with markedly different kinetics: conalbumin mRNA sequences reach half maximum by 1.5 hours, whereas ovalbumin mRNA sequences in these complexes reach half maximum at about eight hours. In the analysis in the accompanying Appendix, we show that the immediate increase of conalbumin mRNA sequences in the nuclear RNP may be accounted for by interaction of the hormone receptor complex with a single regulatory site, whereas the delayed increase of ovalbumin mRNA sequences in the RNP may be due to a requirement for interaction of the hormone receptor complex with multiple regulatory sites.     

Resolution and productivity of conalbumin and lysozyme from fresh egg-white loaded at very high flow rate on a 250-mm length CM-HVFM column.

A 250 x 10 mm I.D. column of CM-HVFM, a novel carboxymethyl ion-exchange matrix, has been used as a preparative chromatographic column to separate fresh egg-white protein. When loading a diluted egg-white solution at pH 4.8 ovalbumin was not adsorbed and lysozyme was preferentially adsorbed compared to the conalbumin. As the column loading was increased from 24 to 450 kg m-3 column volume at the superficial velocity of 6.12 m h-1, the lysozyme continued to be absorbed eventually displacing conalbumin. A maximum lysozyme productivity at 16.7 kg m-3 h-1 was achieved at the highest loading. For conalbumin a maximum productivity of 8.8 kg m-3 h-1 occurred at the lower loading of 100 kg m-3. The purities of lysozyme and conalbumin were comparable at a column loading of 450 kg m-3 h-1. The performance of the column was not degraded, neither was the column blocked or channelled despite the high column loading at the high flow-rate.     

Improvements in immunoprecipitation of specific messenger RNA. Isolation of highly purified conalbumin mRNA in high yield

We have described previously procedures for the isolation of specific mRNA employing immunoprecipitation of polysomes. In spite of our success with ovalbumin mRNA in the chicken oviduct, we have had considerable difficulties in applying these same published techniques to the immunopurification of conalbumin mRNA, despite the fact that the chicken oviduct synthesizes up to 10% of protein as conalbumin. Here we describe a number of modifications and refinements which have proved essential in obtaining intact conalbumin mRNA in high purity and high yields. These refinements include: (a) improved purification of conalbumin in order to remove contaminating proteins that result in impure antibodies; (b) improved isolation of specific conalbumin antibody in high yields; (c) improved methods for reducing contamination by non-specific polysomes; (d) improved techniques for isolation of RNA from immunoprecipitates resulting in less degradation and higher recovery of conalbumin mRNA; (E) improved techniques for efficient translation of conalbumin mRNA involving treatment of the RNA with methylmercury prior to translation. We conclude that problems involved in the immunoprecipitation of different mRNAs may differ, and that various refinements in techniques may be required for obtaining highly purified preparations of intact mRNA in high yields.     

A significant lag in the induction of ovalbumin messenger RNA by steroid hormones: a receptor translocation hypothesis.

Although ovalbumin and conalbumin mRNA accumulate in the same tubular gland cells of the chick oviduct in response to estrogen or progesterone treatment, the kinetics of induction are markedly different. Conalbumin mRNA begins to accumulate within 30 min after estrogen administration, whereas there is a lag of approximately 3 hr before ovalbumin mRNA begins to accumulate, as measured by three independent assays. The kinetics of estrogen-receptor binding to chromatin indicate that these sites are saturated within 15 min of estrogen administration to the chicks, demonstrating that the lag is not due to slow uptake of the steroid. Suboptimal doses of estrogen produce the same lag, but the resultant rate of ovalbumin mRNA accumulation is lower than with an optimal dose. Partial induction of ovalbumin mRNA by a low dose of estrogen does not shorten the lag with an optimal dose. With progesteone, there is a lag of about 2 hr before either ovalbumin or conalbumin mRNA begins to accumulate. Treatment of chicks with hydroxyurea shortens the lag for ovalbumin induction with either hormone. Inhibition of protein synthesis with emetine does not prevent the accumulation of either ovalbumin or conalbumin mRNA. With cycloheximide, however, ovalbumin mRNA accumulation can be prevented. The existence of a lag suggests that there are intermediate steps between the binding of steroid receptors to chromatin and the induction of ovalbumin mRNA. There are basically two models to explain these delays in response: one involving the accumulation of an essential intermediate, and the other involving a rate-limiting translocation of steroid receptors from initial nonproductive chromatin-binding sites to productive sites. Several aspects of the kinetics of ovalbumin mRNA induction are more consistent with the latter model.     

Producing a low ovomucoid egg white preparation by precipitation with aqueous ethanol

A novel method for producing a low ovomucoid egg white preparation is proposed. Egg white powder (0.5 g) was dissolved in a 10-fold weight of distilled water and adjusted to pH 5, and ethanol was added to the solution at a final concentration of 20% (v/v). The mixture was vigorously stirred and centrifuged. The precipitate was washed three times with 20% ethanol (6.25 ml each), with about 65% of egg white proteins occurring in the precipitate. The use of ELISA demonstrated that 70% of ovomucoid was recovered from the supernatant fraction. However, functionally important proteins such as ovalbumin, ovotransferrin, and lysozyme still remained in the precipitate. These results may be due primarily to the much higher solubility of ovomucoid in this aqueous ethanol. Food quality evaluation showed that high whippability and foam stability were retained in the low ovomucoid preparation as in its material egg white. This product would thus be applicable as a new processed food for ovomucoid-sensitive allergic patients.     

Meiotic maturation in Xenopus oocytes: a link between the cessation of protein secretion and the polarized disappearance of Golgi apparati

We have studied the relationship between the timing of the late meiotic events that occur during progesterone-induced oocyte maturation, and intracellular protein transport. We have monitored the secretion of chick oviduct proteins from Xenopus laevis oocytes microinjected with polyadenylated mRNA and found that chick ovalbumin and lysozyme are not secreted during the second meiotic metaphase, in contrast to the earlier prophase stage. Maturation had no detectable effect on the glycosylation of ovalbumin, whereas it affected the glycosylation of chick ovomucoid. As maturation proceeded, the Golgi apparati disappeared in a polarized fashion, beginning in the vegetal half. This disappearance coincided temporally and spatially with that of the nuclear envelope. We speculate that Golgi apparatus disappearance and the block in secretion are causally related.     

Precipitation of egg white proteins below their isoelectric points by sodium dodecyl sulphate and temperature.

The precipitating of effect of sodium dodecyl sulphate (SDS) on the egg white proteins ovalbumin, conalbumin and lysozyme was studied at 25 degrees C and at different pH values. The proteins precipitated below their respective isolectric points, provided the detergent to protein ratio was appropriate. The pH profile of precipitation was different for the three proteins reflecting net charge differences. The binding of SDS to the proteins was studied with [35S]-labelled SDS and for ovalbumin a ratio of 21--28 SDS molecules/protein molecule, dependent on the concentration of SDS initially used, seem to be required for precipitation at pH 4.5. This number, however, is dependent on pH and increases with an increased positive net charge of the protein. The precipitating effect of SDS was identical for ovalbumin, conalbumin and lysozyme when compared on a gram to gram basis (0.1--0.15 g SDS/g precipitated protein). The precipitated protein was denatured as measured by differential scanning calorimetry, but was also completely redissolved if pH was increased to above the isoelectric point. The precipitating effecto f SDS was also examined at elevated temperatures. The two-phase systems of the proteins induced by SDS at 25 degrees C were heated from 25 degrees C to 90 degrees C at a rate of 1.25 degrees C/min. The precipitation behaviour was similar for the three proteins upon heating. When the SDS concentration was increased the precipitation curves were transferred towards lower temperatures and the courses of precipitation became less sharp. The synergistic effect of SDS and heat on protein precipitation was differentiated by denaturation measurements and radioactive labelling. The ratio SDS to precipitated protein gradually diminished towards higher temperatures but no purely thermal precipitation was found.     

Avian egg's white ovomucoid as food-allergen for human

Hen eggs are considered as the most common reason of a food allergy in humans. The most important allergens of egg white proteins are as follows: ovomucoid, lysozyme, ovalbumin and ovomucin. Ovomucoid is a Kazal-type protease inhibitor which accounts for about 10% of avian egg white protein. It is a glycoprotein containing 20 through 25% carbohydrates. The molecule of ovomucoid is composed of three homologous domains. All avian ovomucoid domains contain six cysteines in similar location that form three intradomain disulfide bonds. Ovomucoid (Gal d1) is one of the major allergen in hen's egg. It is a glycoprotein comprising 186 amino acids, and it has a molecular weight of 28000 Da and an isoelectric point of 4.1. Ovomucoid has antibacterial activity resulting from its ability to inhibit bacterial proteolytic enzymes crucial for microbial growth. Many studies reveal that ovomucoid is a thermo stable molecule.