Secretion of egg envelope protein ZPC after C-terminal proteolytic
processing in quail granulosa cells
Tomohiro Sasanami
1
, Jianzhi Pan
1
, Yukio Doi
2
, Miki Hisada
3
, Tetsuya Kohsaka
1
, Masaru Toriyama
1
and Makoto Mori
1
1
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Japan;
2
Department of Food Science,
Kyoto Women’s University, Higashiyama, Kyoto, Japan;
3
Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho,
Mishima-gun, Osaka, Japan
In avian species, an egg envelope homologous to the mam-
malian zona pellucida is called the perivitelline membrane.
We have previously reported that one of its components, a
glycoprotein homologous to mammalian ZPC, is synthe-
sized in the granulosa cells of the quail ovary. In the present
study, we investigated the proteolytic cleavage of the newly
synthesized ZPC and the secretion of ZPC from the granu-

acrosome reaction [7]. In humans and hamsters, ZPC
participates in sperm–egg binding, whereas ZPB is the
primary sperm binding protein in pigs and rabbits [6,8,9].
All of the zona pellucida glycoproteins in the mouse are
synthesized coordinately by the oocytes [10], whereas the
granulosa cells also participate in the formation of the zona
pellucida proteins in the rabbit [11]. The amphibian vitelline
envelope is synthesized by the oocytes [12] while a glyco-
protein of fish chorion is produced in the liver and
transported to the ovary by blood circulation [13].
Two major glycoproteins have been identified as compo-
nents of the inner layer of the vitelline membrane in the
avian oviposited eggs, a similar investment
1
to the PL of
follicular oocytes before ovulation: 33 kDa and 175 kDa in
quail [14] and 32 kDa and 183 kDa in hen [15]. The cDNAs
encoding the 33-kDa protein in quail (GenBank Accession
Number; AB012606) and the 32-kDa protein in the chicken
(GenBank Accession Number; D89097) were cloned, and
these proteins were designated as ZPC from the comparison
of deduced amino-acid sequences of the known ZPC. Avian
ZPC was found to be synthesized in the granulosa cells of
the preovulatory follicles [16,17]. Because granulosa cells are
arranged on the surface of the oocyte as a single layer of
cells, their ZPC production provides a beneficial model for
study of the vectorial
2
secretion of the protein.
Nascent proteins translated in the rough endoplasmic

substrates for furin possesses a conserved consensus
amino-acid sequence, Arg–X–Lys/Arg–Arg [19,26].
In the present study, we examined the proteolytic
cleavage of the newly synthesized ZPC (proZPC) during
post-translational modification and the secretion of the
mature ZPC from quail granulosa cells. To achieve this, we
used two inhibitors that affect the secretory process in the
cell: monensin, an inhibitor of intracellular transport of
protein at the level of Golgi apparatus [27], and brefeldin A
(BFA), a specific inhibitor of membrane transport [28,29].
MATERIALS AND METHODS
Animals and tissue preparation
Female Japanese quail, Coturnix japonica, 15–30 weeks
of age (Tokai-Yuki, Toyohashi, Japan), were main-
tained individually under a photoperiod of 14 h light/10 h
dark with light-on at 0500, and provided with water and a
commercial diet (Tokai-Kigyo, Toyohashi, Japan) ad
libitum. Animals were decapitated and the largest preovu-
latory follicles were dissected and transferred to a physio-
logical saline. The granulosa layer was isolated as a sheet of
granulosa cells sandwiched between the PL and the basal
laminae (BL) as described previously [30].
Culture of granulosa cells
The granulosa layer was cut into 10 pieces, each approxi-
mately 8 mm · 8 mm in size. Each piece was placed into
one well of a 24-well culture plate (Falcon Plastics) and
covered with 1 mL RPMI-1640 medium (Gibco BRL). A
stock solution of monensin (10 m
M
; Wako Pure Chemicals)

out as described previously [31], using 12 and 5%
polyacrylamide for resolving and stacking gels, respec-
tively. For separation of low molecular mass proteins,
tricine/SDS/PAGE was performed [32] with 16.5, 10, and
5% polyacrylamide for resolving, spacer, and stacking
gels, respectively. The gels were stained with Coomassie
brilliant blue R 250 or a silver staining kit (Wako Pure
Chemicals).
For Western blotting, proteins separated on SDS/PAGE
were transferred to a poly(vinylidene difluoride) (PVDF)
membrane (Immobilon-P, Millipore) [33]. After reacting
with antiserum, bands were visualized by a chemilumines-
cent technique (Amersham Pharmacia Biotech) using
horseradish peroxidase-conjugated anti-rabbit
3
IgG (Cappel,
Durham, NC, USA) as a secondary antibody.
Determination of the C-terminus of ZPC
ZPC was purified as described previously from the PL of
preovulatory follicles [34]. Aliquots (2 mg protein) separ-
ated by SDS/PAGE were transferred to PVDF membranes.
The band containing approximately 40 nmol ZPC was
digested at 37 °C for 16 h with 400 pmol lysylendopepti-
dase (EC 3.4.21.50, Wako Pure Chemicals) dissolved in
10% acetonitrile buffered at pH 9.0 with 50 m
M
Tris/HCl.
The ZPC digests were fractionated by RP-HPLC (Model
600, Waters) using a 40–60% acetonitrile gradients in
0.1% trifluoroacetic acid at a flow rate of 1.0 mLÆmin

After culturing, the granulosa layer was extracted with ice-
cold RIPA buffer (300 m
M
NaCl, 2% Nonidet P-40, 1%
deoxycholate, 0.2% SDS, 50 m
M
Tris/HCl pH 7.5) at 4 °C
for 16 h. Insoluble constituents were removed by centrifu-
gation at 14 500 g at 4 °C for 20 min and the supernatant
served as granulosa cell extracts.
2224 T. Sasanami et al. (Eur. J. Biochem. 269) Ó FEBS 2002
To prepare the affinity gel, the IgG fractionated from
anti-(proZPC-derived peptide) serum using a HiTrap Pro-
tein A FF affinity column (Amersham Pharmacia Biotech)
was covalently coupled to Protein A Sepharose FF (Amer-
sham Pharmacia Biotech) with dimethylpimelimidate [36].
The granulosa cell extracts were incubated with the affinity
gel for 16 h at 4 °C. After extensive washing, the gel was
eluted with 1% SDS and the effluent containing proZPC-
derived peptide was dried under a stream of nitrogen gas.
The sample was dissolved in Laemmli’s sample buffer [31],
separated by tricine/SDS/PAGE, and the band of proZPC-
derived peptide transferred to PVDF membrane was
applied directly to an automated gas-phase protein
sequencer (Model 492, Applied Biosystems).
Immunohistochemical observation
For localization of proZPC and ZPC, granulosa layers
cultured with monensin or BFA were fixed in Bouin’s
fixative solution and embedded in Paraplast (Wako Pure
Chemicals). Immunohistochemical techniques were as

immunoreactive 43-kDa protein in the cell lysates decreased
in a time-related manner (Fig. 2B). These results suggest
that the 43-kDa protein is the precursor (proZPC) of
35-kDa ZPC.
Effect of monensin on ZPC secretion
Granulosa layers were cultured with increasing concentra-
tions of monensin, and the media and the cell lysates were
subjected to Western blot analysis. Although an intense band
of 35-kDa ZPC was observed in the medium without
inhibitor, a decreased intensity was detected in the medium
supplemented with monensin in a dose-dependent manner
(Fig. 3A). The addition of 400 ngÆmL
)1
monensin com-
pletely abolished ZPC secretion. In contrast, an increase in
the intensity of all of the bands in the cell lysates was observed
with the addition of 160 ngÆmL
)1
of monensin (Fig. 3B).
Thus, monensin inhibits the secretion of ZPC without
interfering with the conversion of proZPC to 35-kDa ZPC.
Effect of BFA on ZPC secretion
We next investigated the effects of BFA on ZPC secretion.
The addition of 50 ngÆmL
)1
BFA caused a decrease in ZPC
contents in the medium, and 100 ngÆmL
)1
BFA completely
abolished ZPC secretion (Fig. 4A). Although 25 ngÆmL

Immunoblots shown are representative of at least three experiments.
Ó FEBS 2002 Proteolytic processing of ZPC in quail granulosa (Eur. J. Biochem. 269) 2225
Fig. 2. Time course of ZPC content in the medium and the cell lysate during 8 h of culture. Granulosa layers were cultured for 0, 2, 4, 6, or 8 h, and
ZPC protein in the medium (A) and in the cell lysate (B) were detected by using anti-ZPC serum. The intensities of bands were quantified and
plotted as arbitrary units. Values are means ± SEM of three independent experiments with triplicate wells.
Fig. 3. Effects of monensin on ZPC secretion. Granulosa layers were cultured with 0, 80, 160, 240, 320, or 400 ngÆmL
)1
monensin for 6 h. The ZPC
protein in the medium (A) and in the cell lysate (B) were detected by using anti-ZPC serum. Values are means ± SEM of three independent
experiments with triplicate wells.
Fig. 4. Effects of BFA on ZPC secretion. Granulosa layers were cultured with 0, 12.5, 25, 50, or 100 ngÆmL
)1
BFA for 6 h. The ZPC protein in the
medium (A) and in the cell lysate (B) were detected by using anti-ZPC serum. Values are means ± SEM of three independent experiments with
triplicate wells.
2226 T. Sasanami et al. (Eur. J. Biochem. 269) Ó FEBS 2002
4970 Da (Fig. 5B), which coincides with the calculated
molecular mass of the fragment ending at Phe360
(4972.6 Da). This was also supported by the fact that the
C-terminal amino acid was determined as Phe by an
automated C-terminal protein sequencer.
Proteolytic processing of proZPC in granulosa cells
In order to investigate the proteolytic processing of proZPC
in the granulosa cells, we raised antiserum against the
tetradeca peptide located on the C-terminal side of Phe360
(Pro376 to Gln389). Anti-(proZPC-derived peptide) serum
reacted with 43-kDa and 94-kDa but not with 35-kDa ZPC
in the cell lysates and in the PL (Fig. 6, panel 3). In
comparison with that of anti-ZPC serum, anti-(proZPC-
derived peptide) serum tended to react well with the 94-kDa

PL. No positive immunostaining was seen when the
granulosa layer was stained with normal rabbit serum
(Fig. 8B). In contrast, anti-(proZPC-derived peptide)
serum showed the localization of proZPC in the peri-
nuclear region of the cells, but not in the PL (Fig. 8C).
This staining displayed a highly polarized pattern, that is,
the staining was restricted at the apical side of the
Fig. 5. C-Terminal sequence analysis of 35-kDa ZPC. (A) Represen-
tative silver staining pattern of ZPC digested with lysylendopeptidase.
Each PVDF membrane electroblotted with 0 (lane 1) or  40 nmol
ZPC (lane 2) was digested by 400 pmol lysylendopeptidase. The
supernatant of each digest was separated by tricine/SDS/PAGE, and
silver stained. (B) MALDI-TOF MS analysis of purified C-terminal
fragment.
Fig. 6. Representative Western blot analysis of proZPC and ZPC in the
granulosa cells and PL. Granulosa cell lysate (Cell) and PL were
detected with anti-ZPC serum (panel 1, 1 : 2000 dilution), anti-ZPC
serum preincubated with vitelline membrane of oviposited eggs (panel
2), anti-(proZPC-derived peptide) serum (panel 3, 1 : 1000 dilution), or
anti-(proZPC-derived peptide) serum preincubated with antigen pep-
tide (panel 4).
Ó FEBS 2002 Proteolytic processing of ZPC in quail granulosa (Eur. J. Biochem. 269) 2227
perinuclear region corresponding to the PL side, but not
basal side apposed to the BL. When the granulosa layer
was cultured without inhibitors, a similar staining pattern
was obtained by anti-(proZPC-derived peptide) serum,
but the amount of immunoreactive material tended to
decrease (Fig. 8D). The granulosa layer cultured with
monensin was shown to swell, and the entire cytoplasm
was stained (Fig. 8E). The addition of BFA caused a

) for 6 h. The proZPC protein in
thecelllysate(0.5lgproteinperlane)was
detected by using anti-(proZPC-derived pep-
tide) serum. Representative of repeated
experiments.
Fig. 8. Immunohistochemical localization of proZPC and ZPC in granulosa layer. Sections of granulosa layer obtained from 0 (A–C) or 6 h of
culture with control medium (D), with 200 ngÆmL
)1
monensin (E) and with 100 ngÆmL
)1
BFA (F) were processed for immunohistochemical
observation using anti-ZPC serum (A), normal rabbit serum (B), or anti-(proZPC-derived peptide) serum (C–F). Representative of repeated
experiments.
2228 T. Sasanami et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Uchida et al. [38] reported that monensin inhibits the
secretion of procollagen and fibronectin from cultured
human fibroblasts. They also showed that this inhibition is
accompanied by the accumulation of procollagen and
fibronectin in the Golgi apparatus [39,40]. Accumulation
of laminin in the Golgi apparatus was also observed in the
monensin-treated rat astrocytes [41]. In our results, monen-
sin inhibits ZPC secretion without disturbing the conversion
of proZPC to ZPC (Fig. 3). On the other hand, monensin
impedes the proteolytic processing of pro-opiomelanocortin
in rat pituitary cells [42]. This might be due to the fact that
proteolytic processing of pro-opiomelanocortin occurs in
the secretary granule [43]. BFA blocks albumin secretion in
rat hepatocyte by inhibiting the protein transport from
RER to the Golgi complex [44]. As an accumulation of
proalbumin in the RER was observed when cells were

protease. On the other hand, mouse ZPC was reported to be
cleaved at the consensus furin cleavage site without further
processing of its C-terminal paired Arg residues [49]. Such
differences in the process of proteolytic processing between
quail and mouse might reflect the marked species differences
in the properties of their ZPC biosynthesis.
Our results demonstrated that ZPC is never secreted in a
precursor form (see Figs 1–4). Williams and Wassarman
[50] reported ) based on a site-directed point mutation
study ) that secretion of mouse ZPC from transfected cells
is dependent on the cleavage at the consensus furin cleavage
site. The truncation of the C-terminal amino acid of
choriogenin, the precursor protein of the component of
chorion in Oryzias latipes, was also reported to be a
prerequisite for formation of the mature protein and its
assembly into chorion [51]. We suggest that the proteolytic
processing of quail proZPC is considered to be, at least in
part, required for ZPC secretion rather than ZPC biosyn-
thesis. The consensus furin cleavage site was found within
the hydrophobic domain near the C terminus in the ZPC of
all mammalian and avian species studied, though the overall
similarity in amino-acid sequence among the distal classes
was relatively low [6,16]. This indicates that intracellular
processing at the furin cleavage site might universally
participate in the formation of mature ZPC from its
precursor.
The immunohistochemical study with anti-(proZPC-
derived peptide) serum showed that immunoreactive
material is present only on the apical side of the perinuclear
region (Fig. 8C). Therefore ZPC might be transported

observed during insulin biosynthesis in pancreatic b cells
[55], which is regarded as an intermediate of proinsulin to
insulin conversion. Because the intensity of the 94-kDa
band is always parallel to 43 kDa, we suggest that the 94-
kDa protein is an oligomeric intermediate of the 43-kDa
proZPC generated during post-translational modification.
In conclusion, our study suggests that newly synthesized
ZPC is proteolytically cleaved at the consensus furin
cleavage site with furin-like protease, and the resulting
two basic residues at the C-terminus are subsequently
trimmed off with carboxypeptidase H-like protease to
generate the mature 35-kDa ZPC prior to secretion. This
process might be a prerequisite event for ZPC secretion and
its incorporation into PL.
ACKNOWLEDGEMENTS
We are grateful to W. J. Schneider (Department of Molecular
Genetics, Institute of Medical Biochemistry, University and Biocenter
Vienna) for his helpful discussion. This work was supported in part by
Ó FEBS 2002 Proteolytic processing of ZPC in quail granulosa (Eur. J. Biochem. 269) 2229
grant-in-aid for scientific research (09660300, 11660280, and 13660284
to M. M.) from the Ministry of Education, Science, Sports, and
Culture, Japan.
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