Ovalbumin in Developing Chicken Eggs Migrates from Egg White to Embryonic Organs while Changing Its Conformation and Thermal Stability

Abstract

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. 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. Major proteins in albumen or egg white of chicken eggs are synthesized in the tubular gland cells of the magnum, from which they are secreted and coat the egg during its passage within the oviduct (1). The most abundant protein in egg white is ovalbumin, occupying about 55% of the total proteins in newly deposited eggs (2Osuga D.T. Feeney R.E. Whittaker J.R. Tannenbaum S.R. Food Proteins. Avi Publishing Co., Westport, CT1977: 209-266Google Scholar). It consists of three isoforms called A1, A2, and A3, differing in the number of bound phosphate residues (3Egelandsdal B. J. Food Sci. 1980; 49: 570-573Crossref Scopus (116) Google Scholar). Ovalbumin does not act as a proteinase inhibitor, although it belongs to the serpin superfamily (4Gettins P. Patston P.A. Schapira M. BioEssays. 1993; 15: 461-467Crossref PubMed Scopus (96) Google Scholar). The native form of ovalbumin is coagulated easily upon heating, but can be converted artificially into a distinct, heat-stable form called S-ovalbumin by in vitroincubation under alkaline conditions (5Smith D.B. Aust. J. Biol. Sci. 1964; 17: 261-270Crossref Google Scholar). Considering the fact that S-ovalbumin also occurs in unfertilized eggs during long storage (6Nguyen L.T.H. Smith D.B. CSIRO Food Res. Q. 1984; 44: 44-48Google Scholar), we assume that the conversion of ovalbumin is important for embryonic development. What is the fate of ovalbumin and other egg white proteins in developing embryos? These proteins have been found to move into yolk and then into the embryo via the yolk sac membrane (7Sugimoto Y. Saito A. Kusakabe T. Hori K. Koga K. Biochim. Biophys. Acta. 1989; 992: 400-403Crossref PubMed Scopus (25) Google Scholar, 8Sugimoto Y. Hanada S. Koga K. Sakaguchi B. Biochim . Biophys . Acta. 1984; 788: 117-123Crossref Scopus (22) Google Scholar). In addition, egg white proteins have been shown to be taken up into the amniotic fluid and then absorbed by the embryo (9McIndoe W.H. J. Embryol. Exp. Morphol. 1960; 8: 47-53Google Scholar, 10Romanoff A.L. Romanoff A.L. The Avian Embryo. 10391110Macmillan Co., New York1960: 209-266Google Scholar, 11Baintner K. Feher G. Dev. Biol. 1974; 36: 272-278Crossref PubMed Scopus (30) Google Scholar). Although the precise mode of utilization by the embryo is not yet known, ovalbumin and some egg white proteins may at least partly be absorbed into the embryo without extensive digestion, because albumen proteins such as ovalbumin and ovomucoid have been detected in the embryonic blood system (12Marshall M.E. Deutch H.F. J. Biol. Chem. 1951; 185: 155-161Google Scholar). In this study, we confirmed that ovalbumin becomes heat stable during migration from egg white to embryonic organs. Examination by circular dichroism (CD) 1The abbreviations used are: CD, circular dichroism; DSC, differential scanning microcalorimetry; MALDI-TOF-MS, matrix-assisted laser desorption ionization-time of flight-mass spectrometry; native PAGE, polyacrylamide gel electrophoresis under nondenaturing conditions; PAGE, polyacrylamide gel electrophoresis; IgG, immunoglobulin; BSA, bovine serum albumin; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; CHES, 2-(cyclohexylamino)ethanesulfonic acid. and differential scanning microcalorimetry (DSC) supported the idea that the heat-stable form of ovalbumin has undergone conformational changes. The present report may be the first to characterize extensively the naturally occurring heat-stable form of ovalbumin. Fertile eggs of the white Leghorn hen were incubated at 38 ± 0.5 °C at 80% relative humidity. The developmental stages during incubation were assigned according to Hamburger and Hamilton (13Hamburger V. Hamilton H.L. J. Morphol. 1951; 88: 49-92Crossref PubMed Scopus (10048) Google Scholar). Hatching occurred on day 21 or at stage 46 (where day 0 denotes stage 1, shortly after oviposition). Egg white and yolk were separately withdrawn as described in a previous report (7Sugimoto Y. Saito A. Kusakabe T. Hori K. Koga K. Biochim. Biophys. Acta. 1989; 992: 400-403Crossref PubMed Scopus (25) Google Scholar). Amniotic fluid was taken carefully with a syringe needle from the amnion after the stage when it began to be apparent; if the fluid was thin, it was concentrated by lyophilization before analysis. Serum was withdrawn from an extra-embryonic blood vessel with a syringe needle, avoiding contamination of other material. Serum specimens were also collected from hatched chicks and adults. All specimens were processed to remove insoluble material (7Sugimoto Y. Saito A. Kusakabe T. Hori K. Koga K. Biochim. Biophys. Acta. 1989; 992: 400-403Crossref PubMed Scopus (25) Google Scholar, 8Sugimoto Y. Hanada S. Koga K. Sakaguchi B. Biochim . Biophys . Acta. 1984; 788: 117-123Crossref Scopus (22) Google Scholar) before using for further experiments. The stable form of ovalbumin, called S-ovalbumin, was induced according to modification of a previous method (14Huntington J. Patston P. Gettins G.W. Protein Sci. 1995; 4: 613-621Crossref PubMed Scopus (55) Google Scholar); 1% aqueous solution of native ovalbumin purified from day 0 egg white (see below) was filtered through a 0.22-μm Millex filter (Millipore) and incubated at 55 °C for 3 days in 50 mmCHES buffer, pH 9.2; under these conditions almost all albumin molecules were expected to assume the S form (6Nguyen L.T.H. Smith D.B. CSIRO Food Res. Q. 1984; 44: 44-48Google Scholar, 14Huntington J. Patston P. Gettins G.W. Protein Sci. 1995; 4: 613-621Crossref PubMed Scopus (55) Google Scholar). The 1% aqueous solutions of crude or purified ovalbumin were heated at 80, 90, or 97 °C for 10 min, cooled on ice, and centrifuged at 10,000 × g for 15 min at 4 °C; the supernatants and/or precipitates were then subjected to further analysis. Protein concentrations were determined by the phenol method as described (15Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). SDS-PAGE using 12% gels and native PAGE using 8% gels were performed according to the methods of Laemmli (16Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar) and Davis (17Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 404-427Crossref PubMed Scopus (15961) Google Scholar), respectively, in 25 mmTris-glycine, pH 8.0, with or without SDS. Coagulants formed after heating were dissolved in the SDS buffer prior to electrophoresis. The gels were stained with Coomassie Brilliant Blue or subjected to Western blotting with polyvinylidene difluoride membranes (Millipore), which were reacted with anti-rabbit goat IgG (see below) previously labeled with peroxidase (18Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). Antiserum was raised against purified native ovalbumin from day 0 egg white using adult male rabbits and verified by Ouchterlony's double diffusion test (19Ouchterlony D. Acta Pathol. Microbiol. Scand. 1949; 26: 507-515Crossref PubMed Scopus (1211) Google Scholar). IgG against ovalbumin was purified by Protein A (Bio-Rad) column chromatography and then by ovalbumin-agarose affinity chromatography. Ovalbumin was purified by the following three steps: 1) precipitation with 50% saturated ammonium sulfate at pH 4.5 followed by solubilization in 50 mmsodium acetate buffer, pH 4.4 (this step was repeated three times); 2) column chromatography with CM-Cellulofine C-200 m (Seikagaku Kogyo) equilibrated with 50 mm sodium acetate buffer, pH 4.4; ovalbumin fractions were eluted with a linear gradient of NaCl from 0 to 0.4 m made up in the same buffer; when only small amounts of protein were available, CM-Cellulofine column chromatography was replaced by affinity column chromatography using anti-ovalbumin IgG agarose (Pharmacia); 3) gel filtration with a column of SW 3000G (Toso) using 50 mm phosphate buffer, pH 7.0. Far-UV CD spectra (180–250 nm) were recorded at 25 °C with a JASCO 720 spectropolarimeter. Samples were at a concentration of 2.0 μm in 50 mm sodium phosphate buffer, pH 7.4. Data were expressed as mean residue ellipticity (degree cm2/dmol) employing a mean residue molecular weight of 111, which was based on the molecular weight of 42,747 and 385 amino acid residues. The predicted degree of secondary structure was calculated by the program SELCON (20Sreeraman N. Woody R.W. Anal. Biochem. 1993; 209: 32-44Crossref PubMed Scopus (946) Google Scholar). DSC was carried out with a Microcal calorimeter VP-DSC at a heating rate of Protein solutions at in mm sodium phosphate buffer, pH were before analysis. analysis was using a under the conditions by the using bovine serum albumin as a In 10 aqueous solution of protein was in a a and by laser The of was in against molecular Purified ovalbumin solutions at a concentration of were incubated with at °C under The amino acid of some of the was analyzed using an were out and extensively with mm sodium phosphate buffer, pH with mm at 4 these were with in and at μm at were The were with goat serum and 1% for to with PBS, and incubated for with anti-ovalbumin IgG in The were extensively with and then incubated for with goat anti-rabbit IgG with as the specimens were stained by of and The concentration was a of A was performed by using the anti-ovalbumin IgG with ovalbumin as a Western blotting was performed to the ovalbumin in the which were with and in of 50 mm buffer, pH mm and mm before electrophoresis. from embryonic J. T. A Scholar) was on 1% gels and to The filter was ovalbumin K. M. A. A. B. P. Nature. 1979; PubMed Scopus Google Scholar) as a was performed as previously Smith Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) using the and the reverse from the ovalbumin K. M. A. A. B. P. Nature. 1979; PubMed Scopus Google Scholar). ovalbumin, the amniotic fluid and the embryonic serum were from developing eggs and subjected to It was to fluid specimens at stages of development. day 10 or stage the amniotic fluid showed protein a that was confirmed to ovalbumin by Western blotting with anti-ovalbumin IgG The was after day 14 or stage The embryonic serum also exhibited a for ovalbumin at the after day 10 or stage Ovalbumin be detected in the serum specimens from newly hatched chicks and chicks prior to not that serum ovalbumin after hen serum was found to ovalbumin as in the of this be of proteins in embryonic serum from developing Coomassie Blue and the Western with anti-ovalbumin IgG for developing in days after Serum was on and after day 10 Hatching occurred on day 21 serum specimens from shortly after and were other the to to that developing eggs heat-stable ovalbumin by specimens before and after first egg white specimens these were expected to be in ovalbumin. heating the specimens showed A1, A2, and on native PAGE these also showed an ovalbumin after Western blotting 3 heating at °C for 10 min, or was 0 of that the egg white on day 0 or stage 1, shortly after ovalbumin which were in the form. study indicated that a up to of day 0 egg white ovalbumin was heat-stable not In the supernatants on days or stages ovalbumin of these material to S-ovalbumin. was the most on days or stages The three ovalbumin isoforms were present after as in 3 and ovalbumin stable to heating at °C for 10 min was detected at some then analyzed the amniotic fluid stage the egg yolk stage and the embryonic serum stage These also protein in three isoforms in the supernatants after heating at or °C for 10 min These were to ovalbumin by Western blotting analysis not egg day 14 amniotic and day embryonic serum were subjected to the for ovalbumin, in the that the naturally occurring heat-stable be the egg white and amniotic fluid after precipitation in 50% ammonium column chromatography was stable ovalbumin and native ovalbumin were from other on the of differential in a linear gradient of NaCl not Although the were not the be calculated from the relative as and for day egg white and day 14 amniotic the other because the serum after ammonium sulfate precipitation ovalbumin was purified with anti-ovalbumin in of it was not to the forms to a of of the heat-stable ovalbumin fractions from egg white and amniotic as as the from was subjected to the step by gel filtration through SW SDS-PAGE followed by Western blotting A and all preparations exhibited a These results showed that the IgG raised for native ovalbumin with stable ovalbumin. The purified ovalbumin preparations are as indicated in In the and occurring heat-stable ovalbumin as as S-ovalbumin heat-stable artificially induced by heating the native at pH were although with native ovalbumin was also further was carried out with the preparations was and some molecular of purified ovalbumin or used in in the rate material of from CD spectra expressed in of total ordered from CD spectra expressed in of total ordered to DSC (see to (see 0 egg egg egg 14 amniotic 14 amniotic 0 egg by the heating of at 55 °C for 3 days at pH from 14 and from 14 and from 14 and from 14 and from 14 and from 14 and from 14 and from 14 and or were or less between the present and previous native ovalbumin, and between the present and previous S-ovalbumin, because of and/or in the rate material of from CD spectra expressed in of total ordered to DSC (see to (see by the heating of at 55 °C for 3 days at pH from 14Huntington J. Patston P. Gettins G.W. Protein Sci. 1995; 4: 613-621Crossref PubMed Scopus (55) Google and P. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in a or were or less between the present and previous native ovalbumin, and between the present and previous S-ovalbumin, because of and/or was heated at 80, 90, or 97 °C for 10 min, and the supernatants and precipitates were analyzed by SDS-PAGE heating at all material in the 1) and precipitates were that ovalbumin was after heating at or 97 a large of in the supernatants In these precipitates were formed 3 and that some fractions were The the results for as a almost all material was in the precipitates upon heating at or °C 3 and while material was in the supernatants and that was in the native form. the same results as by not analyzed for CD purified preparations of stable and native ovalbumin as as artificially induced S-ovalbumin from exhibited was the between the heat-stable ovalbumin or and the native ovalbumin or S-ovalbumin an The and were calculated from the spectra and are in and on less than and that the occurring heat-stable ovalbumin molecules are in less and a of with the native and with the artificially induced S-ovalbumin. These with results using DSC not which indicated that and a at whereas a at °C (see also for a of All three preparations a at The artificially induced S-ovalbumin an a at °C and at and between native ovalbumin and occurring heat-stable ovalbumin. of ovalbumin preparations revealed about at the of not and the same whereas exhibited a differential and the at an of which was not present in the at of which was present in and but with a The of between be to the of a phosphate residue in the stable ovalbumin. the same was at the of and the of be Although the precise between the of size and heat to be the present indicated that the heat-stable ovalbumin from developing eggs from the native in of In addition, the induced S-ovalbumin a from the native form of ovalbumin. were to the and amino acid of the present ovalbumin because they are by an and an acid J. Biochem. PubMed Scopus Google Scholar). to characterize the its after heating at °C for 10 min, and were with under conditions for and the were subjected to All showed the same A of the and the from these preparations an amino acid of is a of ovalbumin, to residues M. J. Biochem. PubMed Scopus Google Scholar). of developing embryonic were for using anti-ovalbumin These be after day 4 or stage and all specimens for ovalbumin, although not for the native and stable results on day or stage when the system has to extensive (13Hamburger V. Hamilton H.L. J. Morphol. 1951; 88: 49-92Crossref PubMed Scopus (10048) Google Scholar). shown in was most in the central nervous although developing and the of which be below) were to some are in the the In was in the to In was in the developing were in the were in and to and carried out with IgG with amounts of ovalbumin. The was also confirmed by Western the results for specimens on day 18 or stage when is almost for ovalbumin were in the of the the was and the was The for the was by than that for the is reminiscent of the in the on the in A. It is that the migration rate of the in SDS-PAGE was to that of ovalbumin in all the The were for ovalbumin shortly after not from the day and as as from the day 18 and was subjected to the analysis and under the conditions under of the indicated the of ovalbumin not may be or ovalbumin in the or The present study showed that an material in the amniotic yolk, and the central nervous system of the It is to be which is serpin as a and with to ovalbumin Biochim. Biophys. Acta. PubMed Scopus Google Scholar), using and to ovalbumin and and showed for anti-ovalbumin IgG and for anti-ovalbumin with the amino acid we that the material detected in embryo was ovalbumin. Although the has been to be to ovalbumin A. D. P. J. PubMed Scopus Google Scholar), the ovalbumin detected in the present study be the of for ovalbumin were in the and other the of ovalbumin found in the developing embryo be egg also showed that other egg white such as and were in embryonic organs. that may be a of egg white proteins to the developing It is that ovalbumin is present in most of an in amounts from to A previous in of embryo with J. Dev. Biol. 26: Scholar) has indicated that ovalbumin can the and without although a large was to amino that at least a of ovalbumin molecules is from egg white to the embryonic the via the amniotic embryonic and egg yolk, which are all in egg white proteins as and in previous (7Sugimoto Y. Saito A. Kusakabe T. Hori K. Koga K. Biochim. Biophys. Acta. 1989; 992: 400-403Crossref PubMed Scopus (25) Google Scholar, 8Sugimoto Y. Hanada S. Koga K. Sakaguchi B. Biochim . Biophys . Acta. 1984; 788: 117-123Crossref Scopus (22) Google Scholar, W.H. J. Embryol. Exp. Morphol. 1960; 8: 47-53Google Scholar, 10Romanoff A.L. Romanoff A.L. The Avian Embryo. 10391110Macmillan Co., New York1960: 209-266Google Scholar, 11Baintner K. Feher G. Dev. Biol. 1974; 36: 272-278Crossref PubMed Scopus (30) Google Scholar, M.E. Deutch H.F. J. Biol. Chem. 1951; 185: 155-161Google Scholar). It is that the amniotic fluid can into with the embryo via an to the the system is ovalbumin was detected in the it is into cells through a Ovalbumin may be by and other In the heat-stable preparations in the present study were to and than native A previous study (14Huntington J. Patston P. Gettins G.W. Protein Sci. 1995; 4: 613-621Crossref PubMed Scopus (55) Google Scholar) also indicated higher of of S-ovalbumin than the native ovalbumin by and a ovalbumin be detected in the that this is in of The of ovalbumin in by may be by such as and its yolk (7Sugimoto Y. Saito A. Kusakabe T. Hori K. Koga K. Biochim. Biophys. Acta. 1989; 992: 400-403Crossref PubMed Scopus (25) Google Scholar, Y. Kusakabe T. S. T. K. M. T. Koga K. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). In have indicated that the of ovalbumin by and was in these described the naturally occurring heat-stable ovalbumin be from the native by column chromatography. method made it to the without heating in stage egg white and in stage 40 amniotic a higher of 90% was for stage 44 egg yolk not and fractions of ovalbumin were to heating at 97 °C for 10 with the that were by CD and DSC for heat-stable ovalbumin, that forms of stable ovalbumin is with the fact that of S-ovalbumin are during upon pH and conditions (6Nguyen L.T.H. Smith D.B. CSIRO Food Res. Q. 1984; 44: 44-48Google Scholar, 14Huntington J. Patston P. Gettins G.W. Protein Sci. 1995; 4: 613-621Crossref PubMed Scopus (55) Google Scholar). ovalbumin may at stages of although to be the ovalbumin molecules detected in the developing are in a heat-stable form or not is at CD indicated that the heat-stable ovalbumin has undergone conformational a in that stable ovalbumin may naturally from native ovalbumin during while its secondary the of the secondary to heat is artificially induced S-ovalbumin exhibited CD and DSC to of native ovalbumin. was also in a previous report (14Huntington J. Patston P. Gettins G.W. Protein Sci. 1995; 4: 613-621Crossref PubMed Scopus (55) Google Scholar). The analysis by indicated that the of stable ovalbumin a in size the of phosphate residues from some of the J. Biochem. 44: PubMed Scopus (22) Google Scholar) also indicated that ovalbumin with residues is present in the embryonic the size which is an of ovalbumin its differential modification by J. Biochem. 44: PubMed Scopus (22) Google Scholar), as by M. T. H. M. Scholar), may have to the developmental in and configuration about the in between the native and heat-stable of ovalbumin is by that the heat-stable ovalbumin preparations were easily converted by to for and other by heating at or °C for 10 Although the of this is this conversion has been for native ovalbumin in repeated using preparations and under heating conditions has been a report the conversion of a native ovalbumin to against by heating at 97 °C; P. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Egg white ovalbumin has also been to into a heat-stable form during storage of unfertilized eggs at 38 °C (5Smith D.B. Aust. J. Biol. Sci. 1964; 17: 261-270Crossref Google L.T.H. Smith D.B. CSIRO Food Res. Q. 1984; 44: 44-48Google Scholar). was in the present when newly unfertilized eggs were at 38 proteins stable to heating at °C for 10 min occurred in egg white after 3 days not the in the supernatants of developing eggs after heating at °C for 10 min were in unfertilized eggs to at 38 °C for up to The conformational in developing eggs may be from the in unfertilized eggs. that egg white ovalbumin into the embryonic by changing its form to less ordered to Ovalbumin may not be a storage protein as an amino acid but may also have a in developing It be of to this through extensive of ovalbumin and its are to and H. for to Y. of for DSC to M. of the of for of the and to P. of M. of K. of and N. of for of ovalbumin chicken its and cells for ovalbumin,