Ovalbumin in Developing Chicken Eggs Migrates from Egg White
to Embryonic Organs while Changing Its Conformation and Thermal
Stability*
Yasushi
Sugimoto
§,
Shinya
Sanuki
,
Seiichiroh
Ohsako¶,
Yuichiro
Higashimoto
,
Michio
Kondo
,
Junichi
Kurawaki**,
Hisham R.
Ibrahim
,
Takayoshi
Aoki
,
Takahiro
Kusakabe
, and
Katsumi
Koga
From the
Department of Biochemical Science and
Technology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, ¶ National Institute for
Environmental Studies, Onogawa, Tsukuba, Ibaraki 305-0053,
Department of Chemistry, Faculty of Science and Engineering,
Saga University, Saga 840-8502, ** Department of Chemistry, Faculty of
Science, Kagoshima University, 1-21-24 Korimoto, Kagoshima
890-0065, and 
Laboratory of
Sericulture, Kyushu University Graduate School of Bioresource and
Bioenvironmental Sciences, Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan
 |
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.
 |
INTRODUCTION |
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 (2). It
consists of three isoforms called A1, A2, and
A3, differing in the number of bound phosphate residues
(3). Ovalbumin does not act as a proteinase inhibitor, although it
belongs to the serpin superfamily (4). 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 vitro
incubation under alkaline conditions (5). Considering the fact
that S-ovalbumin also occurs in unfertilized eggs
during long storage (6), 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 (7, 8). In addition,
egg white proteins have been shown to be taken up into the amniotic
fluid and then absorbed by the embryo (9-11). 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
(12).
In this study, we confirmed that ovalbumin becomes heat stable during
migration from egg white to embryonic organs. Examination by circular
dichroism (CD)1 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.
 |
EXPERIMENTAL PROCEDURES |
Starting Material--
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 (13). 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 (7).
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 (7, 8) before using for further experiments.
Induction of S-ovalbumin--
The stable form of ovalbumin,
called S-ovalbumin, was induced according to modification of a previous
method (14); 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 mM
CHES buffer, pH 9.2; under these conditions almost all albumin
molecules were expected to assume the S form (6, 14).
Heat Stability Test--
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 Analyses--
Protein concentrations were determined by
the phenol method as described (15). SDS-PAGE using 12% gels and
native PAGE using 8% gels were performed according to the methods of
Laemmli (16) and Davis (17), respectively, in 25 mM
Tris-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 (18).
Preparation of Antibodies--
Antiserum was raised against
purified native ovalbumin from day 0 egg white using adult male rabbits
and verified by Ouchterlony's double diffusion test (19). IgG against
ovalbumin was purified by Protein A (Bio-Rad) column chromatography and
then by ovalbumin-agarose affinity chromatography.
Purification of Ovalbumin--
Ovalbumin was purified by the
following three steps: 1) precipitation with 50% saturated ammonium
sulfate at pH 4.5 followed by solubilization in 50 mM
sodium 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.
CD Spectroscopy and DSC--
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 (20). DSC was
carried out with a Microcal calorimeter VP-DSC at a heating rate of
1 °C/min. Protein solutions at 1 mg/ml in 20 mM sodium
phosphate buffer, pH 7.4, were degassed before analysis.
Determination of Size Microheterogeneity with a Matrix-assisted
Laser Desorption Ionization-Time of Flight-Mass Spectrometer
(MALDI-TOF-MS)--
This analysis was conducted using a Bruker
MALDI-TOF-MS apparatus (REFLEXII) under the conditions specified by the
manufacturer, using bovine serum albumin as a calibrant. In brief, 10 pmol/µl aqueous solution of protein was embedded in a light-absorbing "matrix" (2,5-dihydroxylbenzoic acid), coated onto a sample plate (target), dried, and excited by laser light. The absolute intensity (putative ion mass) of desorbed proton was plotted in arbitrary units
against molecular weight.
Analysis of Partial Amino Acid Sequence--
Purified ovalbumin
solutions at a concentration of 1 mg/ml were incubated with porcine
pepsin (Sigma) at 37 °C under appropriate conditions. The amino acid
sequence of some of the peptide fragments obtained was analyzed using
an Applied Biosystems 491A sequencer.
Immunohistochemistry--
Embryonic organs were dissected out
and washed extensively with PBS (10 mM sodium phosphate
buffer, pH 7.2, with 154 mM saline) at 4 °C. Then these
were fixed with Bouin's solution, embedded in paraffin, and sectioned
at 5 µm thickness at room temperature. Sometimes whole embryos were
similarly processed. The sections were deparaffinized, blocked with PBS
containing 20% normal goat serum and 1% BSA for 1 h to minimize
nonspecific staining, rinsed with PBS, and incubated for 1 h with
affinity-purified anti-ovalbumin rabbit IgG (10 µg/ml) suspended in
PBS containing 0.1% BSA (BSA/PBS). The sections were washed
extensively with PBS and then incubated for 1 h with 8 µg/ml
horseradish peroxidase-conjugated goat anti-rabbit IgG (Pierce). After
washing with PBS as above, the specimens were stained by addition of
0.05% diaminobentizine and 0.01% H2O2. The
optimum antibody concentration was a dilution of 1:2000. A negative
control was performed by using the anti-ovalbumin rabbit IgG
preincubated with 100-fold commercial ovalbumin (w/w) as a primary
antibody. Western blotting was performed to confirm the ovalbumin
distribution in the organs, which were thoroughly washed with PBS and
homogenized in 5 volumes of 50 mM HEPES buffer, pH 7.4, containing 1 mM phenylmethylsulfonyl fluoride and 1 mM EDTA before electrophoresis.
Northern Blotting and RT-PCR--
RNA isolated from embryonic
organs (21) was separated on 1% formaldehyde-agarose gels and
transblotted to Hybond-N+ (Amersham). The filter was hybridized with
32P-labeled ovalbumin cDNA (22) as a probe. PT-PCR was
performed as previously reported (23) using the forward primer
5'-GCAATTCTAGCCATGGTATACCTGG-3' and the reverse primer
5'-GGATCCTGGCTGAAGAGCTAAACAC-3' designed from the ovalbumin gene
sequence (22).
 |
RESULTS |
Detection of Ovalbumin in Amniotic Fluid and Embryonic
Serum--
To detect ovalbumin, the amniotic fluid and the embryonic
serum were prepared daily from developing eggs and subjected to SDS-PAGE. It was possible to collect fluid specimens at later stages of
development. After day 10 or stage 36, the amniotic fluid showed
several protein bands, including a 45-kDa band that was confirmed to
possess ovalbumin by Western blotting with affinity-purified anti-ovalbumin IgG (Fig. 1). The
immunological signal was strong after day 14 or stage 40. The embryonic
serum also exhibited a signal for ovalbumin at the 45-kDa position
after day 10 or stage 36 (Fig. 2).
Ovalbumin could be hardly detected in the serum specimens from newly
hatched chicks (lane H) and young chicks prior to
sexual maturation (data not shown), indicating that serum ovalbumin
disappears rapidly after hatching (although laying hen serum was found
to contain ovalbumin as seen in lane Ad; the
basis of this phenomenon will be considered elsewhere).

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Fig. 1.
SDS-PAGE of proteins in amniotic fluid from
developing eggs: Coomassie Blue stain (A) and the
Western blots with anti-ovalbumin IgG (B).
Numerals along abscissa stand for developing age
in days after oviposition. Amniotic fluid could be collected on and
after day 7 (stage 31); neither proteins nor ovalbumin signals were
seen until day 9 (stage 35), and the relevant lanes are omitted.
Specimens until day 13 (stage 39) were lyophilized prior to
electrophoresis. All concentrations were adjusted to approximately
0.1% by absorbance at 280 nm. M, molecular weight markers,
from top to bottom, are bovine serum albumin, catalase, and lactate
dehydrogenase (sizes are shown along the right
margin). OA, the purified ovalbumin EWN-0 as a
reference (for abbreviations, see Table I). Arrow indicates
ovalbumin position (45 kDa).
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Fig. 2.
SDS-PAGE of proteins in embryonic serum from
developing eggs: Coomassie Blue stain (A) and the
Western blots with anti-ovalbumin IgG (B).
Numerals along abscissa stand for developing age
in days after oviposition. Serum was collectable on and after day 10 (stage 36). Hatching occurred on day 21 (stage 46). Additionally, serum
specimens from neonates shortly after hatching (lane
H) and female adults (lane Ad) were
analyzed. For other details, see the legend to Fig. 1.
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Detection of Heat-stable Ovalbumin in Developing Eggs--
We
tried to demonstrate that developing eggs contained heat-stable
ovalbumin by analyzing specimens before and after heating. We first
examined egg white specimens since these were expected to be rich in
ovalbumin. Before heating the specimens showed isoform bands
A1, A2, and A3 on native PAGE (Fig.
3A); these also showed an
ovalbumin signal after Western blotting (Fig. 3B). After
heating at 80 °C for 10 min, little or no band was seen
(lane 0 of Fig. 3, C and
D), indicating that the egg white specimen on day 0 or stage
1, shortly after oviposition, contained coagulative ovalbumin molecules, which were thus in the native, heat-unstable form. Detailed
study indicated that a subtle fraction, up to 2%, of day 0 egg white
ovalbumin was heat-stable (data not shown). In contrast, the
supernatants on days 3-18 or stages 20-44 gave strong ovalbumin bands
(lanes 3-18 of Fig. 3, C and
D); thus, these contained noncoagulative material comparable
to S-ovalbumin. Banding intensity was the most marked on days 10-12 or
stages 36-38. The three ovalbumin isoforms were still present after
heating. Moreover, as seen in Fig. 3 (E and F),
ovalbumin stable to heating even at 90 °C for 10 min was detected at
some stages.

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Fig. 3.
Native PAGE of proteins in egg white from
developing eggs: Coomassie Blue stain (A,
C, and E) and the Western blots with
anti-ovalbumin IgG (B, D, and
F). Egg white specimens were directly analyzed
(A and B) or the supernatants recovered after
heating for 10 min at 80 °C (C and D) or at
90 °C (E and F) were analyzed.
Numerals along abscissa stand for developing age
in days. Day 0 represents stage 1, shortly after oviposition, with
little or no material in the supernatants after heating (C,
D, E, and F). Lane
0+ represents day 0 egg white before heating (in
A and B, the same as lane
0). A1, A2,
and A3 indicate ovalbumin isoforms due to
differential modification with phosphate (see text). OA is
defined in the legend to Fig. 1.
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We then analyzed the amniotic fluid (day 14, stage 40), the egg
yolk (day 18, stage 44), and the embryonic serum (day 19, stage 45).
These also gave noncoagulative protein bands in three isoforms in the
supernatants after heating at 80 or 90 °C for 10 min (Fig.
4). These bands were ascertained to
contain ovalbumin by Western blotting analysis (data not shown).

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Fig. 4.
Detection of the heat-stable form of
ovalbumin in amniotic fluid (AMF), embryonic serum,
and yolk of developing eggs: Coomassie Blue stain after native
PAGE. The specimens were analyzed directly (lanes
1) or as the supernatants recovered after heating at
80 °C for 10 min (lanes 2) or at 90 °C for
10 min (lanes 3). EW0 and
EW11 show day 0 (stage 1) egg white and day 11 (stage 37) egg white before heating, respectively, run as references.
For A1, A2, and
A3, see the legend to Fig. 3.
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Purification of the Heat-stable Form of Ovalbumin--
Day 11 egg
white, day 14 amniotic fluid, and day 19 embryonic serum were each
subjected to the purification steps for ovalbumin, in the expectation
that the naturally occurring heat-stable form(s) could be isolated. For
the egg white and amniotic fluid specimens, after precipitation in 50%
ammonium sulfate, DEAE-Cellulofine column chromatography was applied,
wherein stable ovalbumin and native ovalbumin were separated from each
other on the basis of differential elution in a linear gradient of NaCl
(data not shown). Although the peaks were not completely pure, the
stable/native ratios could be calculated from the relative peak areas
as 3:1 and 5:1 for day 11 egg white and day 14 amniotic fluid,
respectively. On the other hand, because the serum specimen contained
little protein, after ammonium sulfate precipitation ovalbumin was
purified with anti-ovalbumin IgG-agarose in place of DEAE-Cellulofine, whereby it was not possible to separate the two forms to obtain a ratio
of stable/native. Each of the heat-stable ovalbumin fractions from egg
white and amniotic fluid, as well as the mixture from serum, was
subjected to the final purification step by gel filtration through SW
3000G. Upon SDS-PAGE followed by Western blotting (Fig. 5, A and B), all
final preparations exhibited a single band. These results showed that
the IgG raised for native ovalbumin cross-reacted with stable
ovalbumin.

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Fig. 5.
SDS-PAGE of the purified ovalbumin
preparations: Coomassie Blue stain (A) and the Western
blots hybridized with anti-ovalbumin IgG (B).
Lanes 1, 2, 3,
4, 5, and 6 stand for commercial
(native) ovalbumin, EWN-0, EWS-11, EWS-11 supernatant after heating at
80 °C for 10 min, AMS-14, and the serum ovalbumin mixture,
respectively. M, markers (see the legend to Fig. 1).
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The purified ovalbumin preparations are abbreviated as indicated in
Table I. In the subsequent experiments,
EWS-11 and AMS-14 (the developmentally occurring heat-stable ovalbumin
preparations) as well as S-ovalbumin (the heat-stable one artificially
induced by heating the native preparation EWN-0 at pH 9.2) were mainly used, although EWN-0 (sometimes together with another native ovalbumin preparation, EWN-11) was also analyzed. No further investigation was
carried out with the preparations whose availability was limited.
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Table I
Abbreviations, recoveries, and some molecular properties of purified
ovalbumin preparations
, undetermined or unabbreviated. Molecular data were more or less
different between the present and previous native ovalbumin, and
between the present and previous S-ovalbumin, probably because of
different sources and/or different experimental conditions.
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Confirmation of the Heat Stability of the Purified Ovalbumin
Preparations--
EWS-11 was heated at 80, 90, or 97 °C for 10 min,
centrifuged, and the supernatants and precipitates were analyzed by
SDS-PAGE (Fig. 6; see the
right half). After heating at 80 °C, all
material remained in the supernatant (lane 1) and
precipitates were scarcely formed, indicating that ovalbumin was
stable. Even after heating at 90 or 97 °C, a large part of materials
remained in the supernatants (lanes 2 and
4, respectively). In these cases, however, precipitates were
formed (lanes 3 and 5), indicating
that some fractions were unstable. The left half
of Fig. 6 illustrates the results for EWN-0 as a reference; almost all
material was recovered in the precipitates upon heating at 80 or
90 °C (lanes 3 and 5, respectively) while no material was seen in the supernatants (lanes
2 and 4), confirming again that EWN-0 was in the
native form. AMS-14 gave exactly the same results as those produced by
EWS-11 (data not shown).

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Fig. 6.
SDS-PAGE of the purified ovalbumin
preparations after heating at different temperatures for 10 min:
Coomassie Blue stain. Right half,
EWS-11. Lane 1, supernatant after heating at
80 °C (no precipitate was formed); lanes 2 and
3, supernatant and precipitate, respectively, after heating
at 90 °C; lanes 4 and 5,
supernatant and precipitate, respectively, after heating at 97 °C.
Left half, EWN-0. Lane 1,
analyzed without heating; lanes 2 and
3, supernatant and precipitate, respectively, after heating
at 80 °C; lanes 4 and 5,
supernatant and precipitate, respectively, after heating at 90 °C.
M, markers (see the legend to Fig. 1).
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CD Spectra and DSC of the Heat-stable Ovalbumin
Preparations--
When analyzed for CD spectra, purified preparations
of stable and native ovalbumin as well as artificially induced
S-ovalbumin from EWN-0 exhibited different patterns (Fig.
7). Particularly noticeable was the
difference between the heat-stable ovalbumin (EWS-11 or AMS-14) and the
native ovalbumin (EWN-0 or EWN-11). S-ovalbumin gave an intermediate
curve. The
-helix and
-sheet contents were calculated from the
spectra and are summarized in Table I. EWS-11 and AMS-14 had on average
25% less
-helix than EWN-0 and EWN-11. We conclude that the
developmentally occurring heat-stable ovalbumin molecules are different
in conformation, i.e. less
-helix and a relatively
increased share of
-sheet compared with the native counterparts, and
even with the artificially induced S-ovalbumin.

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Fig. 7.
CD spectra analyzed for the purified
ovalbumin preparations and S-ovalbumin. Mean residue ellipticity
was plotted against the wavelength. Five determinations were made for
each sample, and the spectrum represents the average. , EWN-0; ×,
EWN-11; , S-ovalbumin (induced from EWN-0 at pH 9.2); , EWS-11;
, AMS-14. The -sheet and -helix contents were calculated, and
the results are listed in Table I.
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These conclusions coincided well with our results using DSC (data not
shown), which indicated that both EWS-11 and AMS-14 gave a major
transition at 86.3 °C, whereas EWN-0 gave a major transition at
78.5 °C (see also Table I for a summary of temperature data). All
three preparations gave a shoulder at 82.6 °C. The artificially
induced S-ovalbumin gave again an intermediate pattern (with a peak at
82.6 °C and shoulders at 78.5 and 86.3 °C) between native
ovalbumin and developmentally occurring heat-stable ovalbumin.
Size Microheterogeneity of the Heat-stable Ovalbumin
Preparations--
MALDI-TOF-MS analyses of ovalbumin preparations
revealed about 20 major peaks at the Mr of
44,000-45,000 (data not shown). EWS-11 and AMS-14 shared the same
curve, whereas EWN-0 exhibited a differential curve. EWS-11 and AMS-14
gave the highest peak at an Mr of 44,490, which
was not present in EWN-0. EWN-0 had the highest peak at an
Mr of 44,570, which was present in EWS-11 and
AMS-14 but with a decreased height. The gap of 80 between these
Mr data might be attributable to the loss of a
phosphate residue in the stable ovalbumin. Exactly the same situation
was seen at the peaks of 44,250 and 44,330; again, the difference of 80 might be explained similarly. Although the precise relationship between
the change of size and heat stability remains to be clarified, the
present data clearly indicated that the heat-stable ovalbumin from
developing eggs differed from the native conformer in composition of
subcomponents. In addition, the induced S-ovalbumin gave a pattern
indistinguishable from the native form of ovalbumin.
Partial Amino Acid Sequence of the Heat-stable Ovalbumin
Preparations--
We were unable to analyze directly the N- and
C-terminal amino acid sequences of the present ovalbumin preparations,
probably because they are blocked by an acetylated glycine and an imino acid proline, respectively (24). Therefore, to characterize the peptide
composition, EWN-0, EWS-11, its supernatant after heating at 80 °C
for 10 min, and AMS-14 were each digested with pepsin under conditions
appropriate for each preparation and the products were subjected to
SDS-PAGE. All showed the same peptide patterns (not shown). A mixture
of the fastest and the slowest bands from these preparations gave an
amino acid sequence of
Ala-Ala-Ser-Val-Ser-Glu-Glu-Phe-Arg-Ala-Asp-His-Pro-Phe-Leu-Phe-X-Ile-Lys-His-Ile-Ala-Thr-Asn. This is a bona fide sequence of ovalbumin, corresponding to residues 351-374 (25).
Detection of Ovalbumin in Embryonic Organs--
Thin sections of
developing embryonic organs were examined for immunohistochemical
staining using affinity-purified anti-ovalbumin IgG. These experiments
could be conducted after day 4 or stage 24, and all specimens until
hatching gave positive signals for ovalbumin, although not
discriminated for the native and stable forms. Fig.
8 illustrates typical results on day 5.5 or stage 28, when the brain system has begun to exhibit extensive
organogenesis (13). As shown in panel A,
immunostain was most intensive in the central nervous system, although
mesenchymal tissues, developing kidney, epidermis, and inner
masses (probably the contents of alimentary canal, which will be
considered again below) were to some extent positive. In
panel B, signals are seen in the ependymal tissue
surrounding the ventricle (V). In panel
D, heavy stain was produced in the rhombencephalon (next to
another ventricle, V). In panel F,
stain was clearly detectable in the developing spinal ganglia
(arrowheads). Positive signals were mostly seen in the
cytoplasm. There were no positive reactions in controls (panels C, E, and G,
corresponding to B, D, and F,
respectively), carried out with IgG preincubated with excess amounts of
commercial ovalbumin.

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Fig. 8.
Immunohistochemical detection of ovalbumin in
the chick embryo. Panel A shows a transverse
section of a whole embryo on day 5.5 after oviposition (stage 28),
immunostained with anti-ovalbumin IgG. Areas I,
II, and III in panel A are
the ependyma of hemisphere, rhombencephalon or metacephalon, and spinal
ganglia, which are magnified in panels B,
D, and E, respectively. The control was made
using a serial section immunostained with pre-absorbed IgG with
commercial ovalbumin, and panels C, E,
and G show corresponding regions to B,
D, and F, respectively. The arrowheads
in F show intense signals which are not seen in
G. V, ventricle. Scale
bar = 200 µm in panel A and 40 µm in panels B-D.
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The organ distribution was also confirmed by Western blotting. Fig.
9 illustrates the results for specimens
on day 18 or stage 44, when embryogenesis is almost complete. Positive
signals for ovalbumin were seen in the extracts of many organs
including the head, eye, heart (although the signal was weak), liver
(showing rather complex banding patterns), intestine, spinal cord,
muscle, dermis, and bone (again the signal was weak). The band
intensity for the stomach contents was by far stronger than that for
the stomach tissue itself. This is reminiscent of the positive stain in
the inner masses on the whole mount thin section presented in Fig.
8A. It is noteworthy that the migration rate of the major band in SDS-PAGE was roughly similar to that of control ovalbumin in
all organ extracts including the stomach contents (Fig. 9). The neonate
organs were no longer positive for ovalbumin shortly after hatching
(data not shown).

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Fig. 9.
SDS-PAGE of the extracts of embryonic organs
followed by Western blotting using anti-ovalbumin IgG. Organs were
dissected from embryos on day 18 (stage 44) and, before extraction,
washed extensively with 10 volumes of PBS more than five times until no
extra-organic ovalbumin was detected. Arrow indicates
ovalbumin position (45 kDa).
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Attempt to Detect Ovalbumin mRNA in Embryonic Organs--
RNA
extracted from the day 7 (stage 31) head and carcass as well as from
the day 18 (stage 44) head, muscle, and liver was subjected to the
Northern blot analysis and RT-PCR under the conditions specified under
"Experimental Procedures." None of the extracts indicated the
presence of ovalbumin sequence (data not shown). Thus, there may be
little or no ovalbumin gene expression in the relevant organs or tissues.
 |
DISCUSSION |
The present study showed that an ovalbumin-like material
accumulates in the amniotic fluid, serum, yolk, and organs including the central nervous system of the chick embryo. It is unlikely to be
Hsp47, which is another non-inhibitory serpin functioning as a collagen
chaperon and with 41% homology to ovalbumin (26), since immunological
experiments using poly- and monoclonal antibodies to ovalbumin and
Hsp47 reveal no
cross-reactivity.2 Thin
sections and organ extracts showed positive signals for anti-ovalbumin
IgG (present study) and for anti-ovalbumin monoclonal antibodies.2 Together with the partial amino acid sequence
data obtained here, we conclude that the material detected in embryo
was authentic ovalbumin.
Although the chick liver has been reported to be able to express
ovalbumin (27), the ovalbumin molecule detected in the present study
cannot be the product of de novo synthesis, since no signals
for ovalbumin mRNA were seen in the chick liver and other tissues.
Thus, the major origin of ovalbumin found in the developing embryo must
be egg white. Our preliminary studies also showed that other egg white
members such as lysozyme and ovotransferrin were detectable in
embryonic organs. This suggests that there may be a direct flow of egg
white proteins to the developing embryo.
It is likely that ovalbumin is present in most regions of an embryo,
exhibiting differences in apparent amounts from organ to organ. A
previous in vitro culture of embryo explants with 11-13
somites (28) has indicated that radiolabeled exogenous ovalbumin can
reach the brain and trunk without degradation, although a large part
was digested to amino acids. We propose that at least a fraction of
ovalbumin molecules is transported intact from egg white to the
embryonic organs, including the brain, probably via the amniotic fluid,
embryonic serum, and egg yolk, which are all rich in egg white proteins
as reported here and in previous articles (7-12). It is noteworthy
that the amniotic fluid can come into contact directly with the embryo
via an open channel to the head region, where the neural system is
formed.3 As ovalbumin was
detected in the cytoplasm, it is probably incorporated into cells
through a specific receptor.
Ovalbumin may be digested by chymotrypsin and other proteinases. In
particular, the heat-stable preparations obtained in the present study
were more susceptible to chymotrypsin and thermolysin than native
ovalbumin.2 A previous study (14) also indicated higher
rates of degradation of S-ovalbumin than the native ovalbumin by
porcine pancreatic elastase and subtilisin Carlsberg. However, a 45-kDa
ovalbumin band could be detected in the stomach contents, indicating
that this molecule is rather intact in terms of size. The degradation of ovalbumin in ovo by endogenous proteinases may possibly
be repressed by specific inhibitors such as ovoinhibitor and its yolk
analog named vitelloinhibitor (7, 29). In fact, our preliminary
experiments have indicated that the degradation of ovalbumin by
chymotrypsin and trypsin was effectively inhibited in vitro
by these inhibitors.
As described above, the naturally occurring heat-stable ovalbumin could
be separated from the native one by column chromatography. This method
made it possible to calculate the stable/total ratios without heating
(75% in stage 37 egg white and 83% in stage 40 amniotic fluid).
Moreover, a higher value of 90% was obtained for stage 44 egg yolk
(details not shown), and fractions of ovalbumin were tolerant to
heating even at 97 °C for 10 min. This fact, together with the
finding that complex patterns were obtained by CD and DSC analyses for
heat-stable ovalbumin, suggests that different forms of stable
ovalbumin coexist. This is compatible with the fact that many
intermediary types of S-ovalbumin are obtained during artificial
induction depending upon pH and temperature conditions (6, 14). Thus,
ovalbumin may participate differently at different stages of
development, although details remain to be elucidated. Whether the
ovalbumin molecules detected in the developing organs are in a
heat-stable form or not is uncertain at present.
CD spectroscopy indicated that the heat-stable ovalbumin has undergone
conformational changes including a decrease in
-helix content. We
suppose that stable ovalbumin may arise naturally from native ovalbumin
during development while converting its secondary conformation.
However, the direct relationship of the secondary structures to heat
stability is unclear, since artificially induced S-ovalbumin exhibited
CD and DSC values rather similar to those of native ovalbumin. This was
also observed in a previous report (14).
The current analysis by MALDI-TOF-MS indicated that the formation of
stable ovalbumin accompanies a fluctuation in size microheterogeneity, probably including the removal of phosphate residues from some of the
subcomponents. Earlier work (30) also indicated that ovalbumin with
decreased phosphorus residues is present in the embryonic serum.
Therefore, the size microheterogeneity, which is an intrinsic property
of ovalbumin reflecting its highly differential modification by
phosphorylation (30), as well by glycosylation (31), may have
relevance to the developmental changes in both stability and
configuration reported here.
Additional evidence about the difference in properties between the
native and heat-stable conformers of ovalbumin is provided by our
preliminary observations that the heat-stable ovalbumin preparations
isolated here were easily converted by themselves to potent inhibitors
for chymotrypsin and other proteinases by heating at 80 or 90 °C for
10 min. Although the physiological significance of this finding is
unclear, this conversion has never been observed for native ovalbumin
in repeated trials using different preparations and under variable
heating conditions (although there has been a report describing the
conversion of a certain commercially available native ovalbumin
preparation to "I-ovalbumin" having inhibitory
activity against serine proteinases by heating at
97 °C; see Ref. 32).
Egg white ovalbumin has also been known to change spontaneously into a
heat-stable form during storage of unfertilized eggs at 38 °C (5,
6). This was reproducible in the present study; when newly deposited,
unfertilized eggs were kept at 38 °C, proteins stable to heating at
80 °C for 10 min occurred in egg white after 3 days (data not
shown). However, the bands appearing in the supernatants of developing
eggs after heating at 90 °C for 10 min were never observable in
unfertilized eggs allowed to stand at 38 °C for up to 19 days. The
conformational alteration taking place in developing eggs may therefore
be somewhat different from the spontaneous one seen in unfertilized eggs.
We propose that egg white ovalbumin moves into the embryonic organs by
changing its form to less ordered structures fitted to transportation.
Ovalbumin may not merely be a storage protein serving as an amino acid
source but may also have a more dynamic function in developing organic
cells. It will be of importance to verify this inference through
extensive analyses of ovalbumin and its related proteins.
 |
ACKNOWLEDGEMENTS |
We are very grateful to C. Kawano, R. Kobayashi, and H. Uetsuji for ardent technical assistance; to Y. Hirohata of Sibel Hegner Japan Co. for performing DSC analysis; to Dr.
M. R. Goldsmith of the University of Rhode Island for critical
reading of the manuscript; and to Dr. P. Chambon of Université
Louis Pasteur, Dr. M. Hirose of Kyoto University, Dr. K. Nagata of
Kyoto University, and Dr. N. Eto of Miyazaki University for generous
gifts of ovalbumin cDNA, chicken Hsp47, its monoclonal antibodies,
and hybridoma cells producing monoclonal antibodies for ovalbumin, respectively.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid for
scientific research by the Ministry of Education, Science and Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel./Fax:
81-99-285-8656; E-mail: yasushi{at}chem.agri.kagoshima-u.ac.jp.
2
Y. Sugimoto, unpublished data.
3
S. Ohsako, unpublished data.
 |
ABBREVIATIONS |
The 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.
 |
REFERENCES |
-
Burley, R. W., and Vedehra, D. V.
(eds)
(1989)
The Avian Egg Chemistry and Biology, pp. 129-145, John Wiley & Sons, Inc., New York
-
Osuga, D. T.,
and Feeney, R. E.
(1977)
in
Food Proteins (Whittaker, J. R., and Tannenbaum, S. R., eds), pp. 209-266, Avi Publishing Co., Westport, CT
-
Egelandsdal, B.
(1980)
J. Food Sci.
49,
570-573
-
Gettins, P.,
Patston, P. A.,
and Schapira, M.
(1993)
BioEssays
15,
461-467[Medline]
[Order article via Infotrieve]
-
Smith, D. B.
(1964)
Aust. J. Biol. Sci.
17,
261-270
-
Nguyen, L. T. H.,
and Smith, D. B.
(1984)
CSIRO Food Res. Q.
44,
44-48
-
Sugimoto, Y.,
Saito, A.,
Kusakabe, T.,
Hori, K.,
and Koga, K.
(1989)
Biochim. Biophys. Acta
992,
400-403[Medline]
[Order article via Infotrieve]
-
Sugimoto, Y,
Hanada, S.,
Koga, K.,
and Sakaguchi, B.
(1984)
Biochim. Biophys. Acta
788,
117-123
-
McIndoe, W. H.
(1960)
J. Embryol. Exp. Morphol.
8,
47-53
-
Romanoff, A. L.
(1960)
in
The Avian Embryo (Romanoff, A. L., ed), pp. 209-26610391110Macmillan Co.New York
-
Baintner, K.,
and Feher, G.
(1974)
Dev. Biol.
36,
272-278[Medline]
[Order article via Infotrieve]
-
Marshall, M. E.,
and Deutch, H. F.
(1951)
J. Biol. Chem.
185,
155-161
-
Hamburger, V.,
and Hamilton, H. L.
(1951)
J. Morphol.
88,
49-92
-
Huntington, J.,
Patston, P.,
and Gettins, G. W.
(1995)
Protein Sci.
4,
613-621[Abstract/Free Full Text]
-
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275[Free Full Text]
-
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
-
Davis, B. J.
(1964)
Ann. N. Y. Acad. Sci.
121,
404-427[Medline]
[Order article via Infotrieve]
-
Towbin, H.,
Staehelin, T.,
and Gordon, J.
(1979)
Proc. Natl. Acad. Sci. U. S. A.
76,
4350-4354[Abstract]
-
Ouchterlony, D.
(1949)
Acta Pathol. Microbiol. Scand.
26,
507-515
-
Sreeraman, N.,
and Woody, R. W.
(1993)
Anal. Biochem.
209,
32-44[CrossRef][Medline]
[Order article via Infotrieve]
-
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Gannon, F.,
O'Hare, K.,
Perrin, F.,
LePennec, J. P.,
Benoist, C.,
Cochet, M.,
Breathnach, R.,
Royal, A.,
Garapin, A.,
Cami, B.,
and Chambon, P.
(1979)
Nature
278,
428-434[Medline]
[Order article via Infotrieve]
-
Kawasaki, E. S.,
Clark, S. S.,
Coyne, M. Y.,
Smith, S. D.,
Champlin, R.,
Witte, O. N.,
and McCormick, F. P.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
5698-5702[Abstract]
-
Nisbet, A. D.,
Saundry, R. H.,
Moir, A. J. G.,
Fothergill, L. A.,
and Fothergill, J. E.
(1981)
Eur. J. Biochem.
115,
335-345[Abstract]
-
Tatsumi, E.,
and Hirose, M.
(1997)
J. Biochem. (Tokyo)
122,
300-308[Abstract]
-
Clarke, E. P.,
and Sanwal, B. D.
(1992)
Biochim. Biophys. Acta
1129,
246-248[Medline]
[Order article via Infotrieve]
-
Dierich, A.,
Gaub, M.-P.,
LePennec, J.-P.,
Astinotti, D.,
and Chambon, P.
(1987)
EMBO J.
6,
2305-2312[Abstract]
-
Hassel, J.,
and Klein, N. W.
(1971)
Dev. Biol.
26,
3880-392
-
Sugimoto, Y.,
Kusakabe, T.,
Nagaoka, S.,
Nirasawa, T.,
Tasuguchi, K.,
Fujii, M.,
Aoki, T.,
and Koga, K.
(1996)
Biochim. Biophys. Acta
1295,
96-102[Medline]
[Order article via Infotrieve]
-
Saito, Z.,
and Martin, W. G.
(1966)
Can. J. Biochem.
44,
293-301[Medline]
[Order article via Infotrieve]
-
Koketsu, M.
(1997)
in
Hen Eggs (Yamamoto, T., Tuneja, L. R., Hatla, H., and Kim, M., eds), pp. 99-115, CRC Press, Boca Raton, FL
-
Mellet, P.,
Michels, B.,
and Bieth, J. G.
(1996)
J. Biol. Chem.
271,
30311-30314[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.