Thermostabilization of ovalbumin by an alkaline treatment: examination for the possible implications of an altered serpin loop structure

Ovalbumin, a member of the serpin superfamily, is transformed via an intermediate state into a non-cleaved, thermostabilized form (S-ovalbumin) during either the storage of unfertilized eggs or development of fertilized eggs; essentially the same thermostabilization also occurs upon in vitro incubation of isolated ovalubumin under alkaline conditions. To investigate the implications of a partial insertion of the alpha-helical serpin loop into beta-sheet A that has been proposed as a conformational mechanism for S-ovalbumin production, we examined the thermostabilization process of ovalbumin with different loop structures. When the thermostabilization processes were compared for the intact, P1-P1'-cleaved and P1-P1'/P8-P7-cleaved forms of egg white ovalbumin, both the rates for the conversion from the native to intermediate and from the intermediate to S-ovalbumin were almost indistinguishable among the three protein forms. Furthermore, the fully loop-inserted form of recombinant ovalbumin mutant R339T that had been thermostabilized by P1-P1' cleavage with Tm values from 72 to 88 degrees C was further thermostabilized by an alkaline treatment, yielding a final product (loop inserted S-ovalbumin) with a Tm value of 93 degrees C. No significant difference was found between native ovalbumin and S-ovalbumin in respect of the rate of proteolytic cleavage of the loop by elastase and subtilisin. These data strongly suggest that S-ovalbumin is produced by a mechanism other than that of the partial loop insertion model.     

Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability

Ovalbumin was detected in developing chicken eggs. The large majority of these ovalbumin molecules was found to be in a heat-stable form reminiscent of S-ovalbumin. About 83 and 90% of the ovalbumin population was in a heat-stable form in day 14 or stage 40 amniotic fluid and day 18 or stage 44 egg yolk, respectively, whereas ovalbumin in newly deposited eggs was in the heat-unstable, native form. Purified preparations of stable ovalbumin from egg white and amniotic fluid showed a less ordered configuration than native ovalbumin, as analyzed by circular dichroism and differential scanning calorimetry. In addition, mass spectrometric analysis exhibited distinct size microheterogeneity between the stable and native forms of ovalbumin. Immunohisotochemical study revealed that ovalbumin was present in the central nervous system and other embryonic organs. These results indicated that egg white ovalbumin migrates into the developing embryo while changing its higher order structure.     

Structural properties of recombinant ovalbumin and its transformation into a thermostabilized form by alkaline treatment.

The recombinant ovalbumin produced in Escherichia coli was purified from the cytoplasmic fraction and analyzed for its chemical and conformational properties. The recombinant ovalbumin displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as egg white ovalbumin. As in the egg white protein, four cysteine sulfhydryls and one cystine disulfide were contained in the recombinant protein, according to the results of amino acid analyses; the disulfide bond was found by a peptide mapping analysis to correspond to the native cystine, Cys73-Cys120. According to a gel electrophoresis analysis, the presence of the disulfide bond was accounted for by specific oxidation of the corresponding cysteine residues during purification of the cytoplasmic protein. Unlike the identity in the conformational and peptide structures, none of the post-translational modifications (N-terminal acetylation, phosphorylation, and glycosylation) that are known with egg white ovalbumin were detected in the recombinant protein. The recombinant ovalbumin was transformed into a thermostabilized form in a similar manner to the transformation of egg white protein into S-ovalbumin; alkaline treatment increased the temperature for thermostability by 8.7 degrees C. These data strongly suggest that the post-translational modifications of ovalbumin are not related to the formation mechanism for S-ovalbumin.     

Thermostability of refolded ovalbumin and S-ovalbumin

Ovalbumin, a member of the serpin superfamily, is transformed into a thermostabilized form, S-ovalbumin, during storage of shell eggs or by an alkaline treatment of the isolated protein (DeltaT(m)=8 degrees C). As structural characteristics of S-ovalbumin, three serine residues (Ser164, Ser236 and Ser320) take the D-amino acid residue configuration, while the conformational change from non-thermostabilized native ovalbumin is very small. To assess the role of the structural characteristics on protein thermostabilization, ovalbumin and S-ovalbumin were denatured to eliminate the conformational modulation effects and then refolded. The denatured ovalbumin and S-ovalbumin were correctly refolded into the original non-denatured forms with the corresponding differential thermostability. There was essentially no difference in the disulfide structures of the native and refolded forms of ovalbumin and S-ovalbumin. These data are consistent with the view that the configuration inversion, which is the only chemical modification directly detected in S-ovalbumin so far, plays a central role in ovalbumin thermostabilization. The rate of refolding of S-ovalbumin was greater than that of ovalbumin, indicating the participation, at least in part, of an increased folding rate for thermodynamic stabilization.     

Deglycosylation of ovalbumin prohibits formation of a heat-stable conformer

To study the influence of the carbohydrate-moiety of ovalbumin on the formation of the heat-stable conformer S-ovalbumin, ovalbumin is deglycosylated with PNGase-F under native conditions. Although the enzymatic deglycosylation procedure resulted in a complete loss of the ability to bind to Concavalin A column-material, only in about 50% the proteins lost their complete carbohydrate moiety, as demonstrated by mass spectrometry and size exclusion chromatography. Thermal stability and conformational changes were determined using circular dichroism and differential scanning calorimetry and demonstrated at ambient temperature no conformational changes due to the deglycosylation. Also the denaturation temperature of the processed proteins remained the same (77.4 +/- 0.4 degrees C). After heat treatment of the processed protein at 55 degrees C and pH 9.9 for 72 h, the condition that converts native ovalbumin into the heat-stable conformer (S-ovalbumin), only the material with the intact carbohydrate moiety forms this heat-stable conformer. The material that effectively lost its carbohydrate moiety appeared fully denatured and aggregated due to these processing conditions. These results indicate that the PNGase-F treatment of ovalbumin prohibits the formation and stabilization of the heat-stable conformer S-ovalbumin. Since S-ovalbumin in egg protein samples is known to affect functional properties, this work illustrates a potential route to control the quality of egg protein ingredients.     

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

Proteomic analysis of egg white proteins during storage

Egg storage causes egg white to lose its viscous nature to form a thin liquid, commonly referred to as egg white thinning. To understand the mechanisms underlying egg white thinning, white-shell eggs were used in the present study to determine the proteome-level changes of egg white proteins occurred during storage. Egg white thinning was observed visually after 20 days of storage at ambient temperature (22 ± 2 °C) when the maximum number of proteome-level changes occurred. The proteins that showed significant changes in abundance during storage included ovalbumin, clusterin, ovoinhibitor, ovotransferrin, and prostaglandin D2 synthase. Among these, only the abundance of clusterin was observed to change continuously during the storage period. Hence, it is expected that the increase in the concentrations of clusterin and ovoinhibitor along with the change of ovalbumin content during storage might contribute to egg white thinning. Degradation of ovalbumin/clusterin during egg storage may be due to the combined effect of proteolysis and increase in pH; this may also be partly responsible for egg white thinning phenomenon.     

Thermostabilized ovalbumin that occurs naturally during development accumulates in embryonic tissues

We have reported that ovalbumin accumulates without digestion in various tissues during embryonic development of the chicken. There are different types of ovalbumin with respect to thermal stability and one of them, which was named "HS-ovalbumin" in the present study, was found to have a T(m) value of 83 degrees C and to be present dominantly in albumen, egg yolk, amniotic fluid, and serum of fertilized eggs. HS-ovalbumin, arising physiologically from its native form (N-ovalbumin), is reminiscent of the previously described intermediate form appearing during the production processes of the so-called S-ovalbumin, which disappeared shortly in fertilized eggs. We showed that HS-ovalbumin is distinguishable from S-ovalbumin by a monoclonal antibody and also from N-ovalbumin by the stability to heating. At the late stages of development, ovalbumin of amniotic fluid seems to be swallowed through pharynx, carried in the intestine through stomach, and absorbed in the blood. Analyses by monoclonal antibody and heat treatment indicated that the HS-form occupies the largest fraction of ovalbumin that accumulates in the embryonic tissues. The current findings suggest that HS-ovalbumin is crucial for embryogenesis.     

Toxic interaction of fumonisin B1 and fusaric acid measured by injection into fertile chicken egg

Toxic interactions of fusaric acid and fumonisin B₁, two mycotoxins produced byFusarium moniliforme, were studied in the chicken embryo. The yolk sacs of fertile White Leghorn eggs were injected before incubation with separate and combined solutions of either fusaric acid and or fumonisin B₁. The toxins were administered in either a sterile 10 mM buffered phosphate solution, pH 6.90, which produced a final pH of 6.6 ± 0.2, or sterile distilled water. Toxicity was based on absence of egg pip at the end of the 21-day incubation period. Toxins administered in the phosphate buffer solution were more toxic than those administered in distilled water. When both toxins were combined in equal concentrations and injected into eggs, increased toxicity resulted. Fusaric acid was shown to be a mild toxin to the eggs and when a relatively nontoxic concentration of it was combined with graded doses of fumonisin B₁, a synergistic toxic response was obtained. Fusaric acid is only moderately toxic to the chicken egg, however its co-occurrence with other fusaria toxins found on corn and other cereals might present possible antagonisms or synergisms. The results of this egg model suggest that fusaric acid might play a role in enhanced and unpredicted toxicity in mammalian systems if it is consumed with other mycotoxins.     

A Raman difference spectroscopic investigation of ovalbumin and S-ovalbumin

Raman spectra from 800 to 1850 cm^?1 of aqueous solutions of ovalbumin and its more heat-stable form, S-ovalbumin, are presented. A Raman difference spectrum (ovalbumin minus S-ovalbumin) shows differences in intensity in the amide I and III regions. These intensity differences lead us to postulate that the conversion of ovalbumin to S-ovalbumin involves a conformation change of a small part (～3–4%) of the protein from α-helix to antiparallel β-sheet geometry. This small difference in the three-dimensional arrangement of the peptide chain may contribute to the large difference in the thermodynamic stability between ovalbumin and S-ovalbumin.     

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.     

Enhanced C-type lysozyme content of wood duck (Aix sponsa) egg white: an adaptation to cavity nesting?

Abstract Wild waterfowl species often nest in conditions where high humidity and microbial contamination may influence egg survival and quality. Albumen is traditionally regarded as the major impediment to microbial contamination of eggs, and its composition and activity may be selected by environmental pressures. Egg white protein from the eggs of wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis), and mute swan (Cygnus olor) was evaluated in order to compare the antimicrobial defenses of these species. Ovotransferrin and ovalbumin were identified in all species, but c-type lysozyme was present only in wood duck and hooded merganser egg white samples. Wood duck egg white showed the greatest bacterial activity as well as the highest lysozyme content. Egg white from wood duck and hooded merganser possessed greater lysozyme activity under acidic conditions, suggesting a c-type lysozyme with a pH optimum lower than that of Gallus gallus c-type lysozyme or the presence of g-type lysozyme. Ovotransferrin bacteriostatic activity appeared to be similar across the species investigated. The results suggest that lysozyme and ovotransferrin play a role in the antimicrobial defense of the avian egg. High levels of the broad-acting c-type lysozyme appear to have evolved in the albumen of the wood duck in order to ensure proper development of the embryo in the humid conditions of the cavity nest.     

Transition of ovalbumin to thermostable structure entails conformational changes involving the reactive center loop

Ovalbumin is a serpin without inhibitory activity against proteases. During embryonic development, ovalbumin in the native (N) form undergoes changes and takes a heat-stable form, which was previously named HS-ovalbumin. It has been known that N-ovalbumin is artificially converted to another thermostable form called S-ovalbumin by heating at an alkaline pH. Here, we characterized further the three ovalbumin forms, N, HS, and S. The epitope of the monoclonal antibody 2B3/2H11, which recognizes N- and HS-ovalbumin but not S-ovalbumin, was found to reside in the region Glu-Val-Val-Gly-Ala-Ser-Glu-Ala-Gly-Val-Asp-Ala-Ala-Ser-Val-Ser-Glu-Glu-Phe-Arg, which corresponds to 340-359 of amino acid residues and is contained in the reactive center loop (RCL). Removal of RCL by elastase or subtilisin mitigated binding of the antibody. Dephosphorylation experiments indicated that the phosphorylated Ser-344 residue located on RCL is crucial for the epitope recognition. We suggest that the shift to the heat-stable form of ovalbumin accompanies a movement of RCL.     

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vibration analysis on incubating eggs and its relation to embryonic development

Coucke (1998) was the first to use acoustic resonance analysis to monitor embryo development in chicken eggs. He remarked that at around 100 hours of incubation, the course of the resonant frequency and damping changed abruptly in the case of fertile eggs. He also showed that these changes were related to a physiologic event during early embryonic development. The objective of our study is to monitor the course of the vibration parameters during the early incubation of chicken eggs and to relate these changes to egg and embryo characteristics. A total of 72 Hybro eggs were incubated vertically in a small incubator at standard conditions. Several egg parameters were measured before incubation. During the early stages of incubation the vibration behavior of these eggs was monitored. The time at which the damping of the vibration suddenly changed, the diameter of the eggs and their interaction were found to be significant explanatory variables in order to predict hatching time. A correlation coefficient r of 0.72 was obtained.     

Crystal structure of S-ovalbumin as a non-loop-inserted thermostabilized serpin form

Ovalbumin, a non-inhibitory member of serine proteinase inhibitors (serpin), is transformed into a heat-stabilized form, S-ovalbumin, under elevated pH conditions. The structural mechanism for the S-ovalbumin formation has long been a puzzling question in food science and serpin structural biology. On the basis of the commonly observed serpin thermostabilization by insertion of the reactive center loop into the proximal beta-sheet, the most widely accepted hypothetical model has included partial loop insertion. Here we demonstrate, for the first time, the crystal structure of S-ovalbumin at 1.9-A resolution. This structure unequivocally excludes the partial loop insertion mechanism; the overall structure, including the reactive center loop structure, is almost the same as that of native ovalbumin, except for the significant motion of the preceding loop of strand 1A away from strand 2A. The most striking finding is that Ser-164, Ser-236, and Ser-320 take the d-amino acid residue configuration. These chemical inversions can be directly related to the irreversible and stepwise nature of the transformation from native ovalbumin to S-ovalbumin. As conformational changes of the side chains, significant alternations are found in the values of the chi 1 of Phe-99 and the chi 3 of Met-241. The former conformational change leads to the decreased solvent accessibility of the hydrophobic core around Phe-99, which includes Phe-180 and Phe-378, the highly conserved residues in serpin. This may give a thermodynamic advantage to the structural stability of S-ovalbumin.     

The presence of heat-stable conformers of ovalbumin affects properties of thermally formed aggregates

The aim of this work was to study the effect of the formation of more heat-stable conformers of chicken egg ovalbumin during incubation at basic pH (9.9) and elevated temperature (55 degrees C) on the protein aggregation properties at neutral pH. Native ovalbumin (N-OVA) is converted on the hours time-scale into more heat-stable forms denoted I- (intermediate) and S-OVA, that have denaturation temperatures 4.8 and 8.4 degrees C, respectively, higher than that of N-OVA. The conversions most likely proceed via I-OVA, but direct conversion of N-OVA into S-OVA with slower kinetics can not be excluded. It is demonstrated that both I- and S-OVA have similar denaturation characteristics to N-OVA, except that higher temperatures are required for denaturation. The presence of even small contributions of I-OVA does, however, reduce the Stokes radius of the aggregates formed upon heat treatment of the material at 90 degrees C about 2-fold. This affects the gel network formation considerably. Since many (commercial) preparations of ovalbumin contain varying contributions of the more heat-stable forms mentioned, proper characterization or standardization of the isolation procedure of the material is essential to control or predict the industrial application of this protein.     

Plasma prolactin levels in incubating turkey hens during pipping of the eggs and after introduction of poults into the nest

Plasma levels of prolactin (Prl) associated with incubation and maternal behavior were compared in turkey hens allowed to incubate 10 fertile eggs (Group I, n = 9) or 10 infertile eggs (Group II, n = 7) in open nest boxes. At the end of the day that the first egg hatched, all unhatched eggs were removed from Group I hens and each hen was given 10 poults. At the end of the following day, infertile eggs were removed from Group II hens and each hen was given 10 poults. Although pipping of the eggs changed the incubation behavior of Group I hens, it had no effect on plasma Prl. Subsequent hatch of the eggs and/or presence of poults resulted, within 24 h, in a sharp fall in Prl levels, abandonment of the nests, and a shift to maternal behavior. Visual and auditory exposure to Group I poults had no effect on plasma Prl or incubation behavior of Group II hens incubating infertile eggs in adjacent pens. However, within 24 h after the infertile eggs were exchanged for newly hatched poults, Prl levels in Group II hens declined sharply and the hens abandoned the nests and showed maternal behavior similar to that observed in Group I hens. No significant relationships were found in either group between plasma Prl levels and quality of incubation or maternal behavior.     

Absorption, transportation and digestion of egg white in quail embryos

The present study was done to reveal how egg white is taken up by embryonic tissues, the pathway through which egg white is transported, and the location where it is digested during the development of the quail Coturnix japonica. Antiserum against quail ovalbumin was raised in rabbit and used as a probe. By immunoelectron microscopy, the uptake of ovalbumin on a small scale by receptor-mediated endocytosis was observed in the ectodermal cells of the yolk sac on days four to seven of incubation. The uptake of egg white on a large scale by fluid-phase endocytosis took place in the cells generally referred to collectively as the 'albumen sac'. The ovalbumin was transported through the albumen sac into the extraembryonic cavity during days eight to 10, and then into the amniotic cavity through the amnion approximately on day 10. Ovalbumin was present in the intestinal lumen on days 11 and 14, but it was not digested in the intestinal epithelial cells. The ovalbumin was detected in the yolk of embryos after day 10. Immunoblot testing, as well as a fluoroimmunoassay, revealed that the location where the amount of ovalbumin was highest changed chronologically from the extraembryonic cavity on day 10 to the amniotic cavity on day 11, the intestinal lumen on day 12 and then to the yolk on day 13. Several low molecular proteins which cross-reacted with the antiserum were observed in the extracts of the yolk. The reaction producing these proteins depended on low pH (approximately 3.0) and was inhibited by pepstatin A. The ovotransferrin was similarly digested. These results indicate that egg white is, for the most part, transported through the albumen sac to the yolk via the extraembryonic cavity, the amniotic cavity, and the intestinal lumen, and is digested in the yolk by aspartic proteinases.     

OSMOTIC RELATIONSHIPS IN THE HEN'S EGG

Data have been given to illustrate the difficulty of obtaining consistent freezing point data with a viscous fluid such as the yolk of the hen''s egg and a technique has been described for obtaining reproducible and accurate results consistently. Further freezing point data have been given which were obtained with both fertile and unfertile hen''s eggs by the use of a freezing point method previously described by the writer. These data show that there is a pronounced difference between the freezing points of the yolk and the white in contrast to data obtained by the use of the same method by Howard who found the freezing points of the yolk and the white to be the same. It was shown by freezing point determinations that even in a mixture of yolk and white osmotic equilibrium is slowly arrived at. This again emphasizes the fact established by Smith and Shepherd that since osmotic equilibrium between yolk and white is slowly arrived at, the postulation of a vital activity at the yolk membrane is unnecessary, since the steady state previously postulated need not be assumed to exist.