SecretionofeggenvelopeproteinZPCafterC-terminal proteolytic
processing inquailgranulosa 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 eggenvelope 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 granulosacellsof the quail ovary. In the present
study, we investigated the proteolytic cleavage of the newly
synthesized ZPC and the secretionofZPC from the granu-
losa cells. Western blot analysis of the cell lysates demon-
strated that the 43-kDa protein is the precursor of mature
ZPC (proZPC), and is converted to the 35-kDa protein
before secretion. The accumulation of proZPC in the pres-
ence of brefeldin A, and conversion of proZPC to ZPC in
the presence of monensin, indicate the possibility that the
proteolytic processingofZPC occurs in the Golgi apparatus.
An analysis of amino-acid sequence identified that the C
terminus of mature ZPCprotein is Phe360, and the N-ter-
minal amino-acid sequence of the proZPC-derived fragment
was determined as Asp363. These results suggest that newly
synthesized ZPC is cleaved at the consensus furin cleavage
site, and the resulting two basic residues at the C terminus are
subsequently trimmed off to generate mature ZPC prior to
secretion.
Keywords: zona pellucida; ZPC; granulosa cell; quail; post-
translational modification.
The plasma membrane of oocytes of all vertebrates is
overlaid with extracellular matrix generally called the egg
envelope, although different names have been adopted for
different classes: zona pellucida for mammals, perivitelline
layer (PL) for birds, vitelline envelope for amphibians, and
chorion for fish. Mouse zona pellucida is composed of three
glycoproteins, ZP1, ZP2, and ZP3 [1], also known as ZPB,
ZPA and ZPC, respectively [2]. For most mammalian
species and other vertebrates, the homologous proteins are
identified in the eggenvelope [2–6].
The eggenvelope plays a significant role in species-
specific sperm–egg interaction. In mice, sperm binds to
O-linked oligosaccharides of ZPC, and undergoes the
acrosome reaction [7]. In humans and hamsters, ZPC
participates in sperm–egg binding, whereas ZPB is the
primary sperm binding proteinin 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 proteininquail (GenBank Accession
Number; AB012606) and the 32-kDa proteinin 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 granulosacells of
the preovulatory follicles [16,17]. Because granulosacells 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
reticulum (RER) receive post-translational modifications
including removal of signal sequence, formation of disulfide
bonds, glycosylation, and proteolytic cleavage. The proteo-
lytic cleavage of the precursor protein is achieved by
digestion by a proprotein convertase, homologous to yeast
subtilisin/kexin that cleaves specific basic amino acid
residues in the substrates [18,19]. So far, seven mammalian
subtilisin/kexin-like proprotein convertases responsible for
intracellular cleavages have been described, including furin,
PC1 (also called PC3), PC2, PC4, PACE4, PC5 (also called
PC6) and PC7 (also called LPC or PC8) [18,20]. PC1 and
PC2 are found in the endocrine and neuroendocrine tissues,
Correspondence to: M. Mori, Department of Applied Biological
Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya,
Shizuoka 422-8529, Japan.
Fax: + 81 54 2384866; E-mail: acmmori@agr.shizuoka.ac.jp
Abbreviations: PL, perivitelline layer; BFA, brefeldin A; RER,
rough endoplasmic reticulum; BL, basal laminae; PVDF,
poly(vinylidene difluoride).
Enzyme: lysylendopeptidase (EC 3.4.21.50).
Note: the GenBank accession number of proteins mentioned in the text
are: 33-kDa proteininquail (quail ZPC), AB012606; 32-kDa protein
in chicken (chicken ZPC), D89097.
(Received 23 October 2001, revised 26 February 2002,
accepted 12 March 2002)
Eur. J. Biochem. 269, 2223–2231 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02880.x
and recognize the paired basic amino acids (Arg–Arg or
Lys–Arg) in the substrates [18,21–23]. Furin is ubiquitously
expressed in all tissues and cell lines examined so far [19,24],
and is localized in the trans-Golgi network [25]. The
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 secretionof the
mature ZPC from quailgranulosa 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 ofgranulosa 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)
and BFA (5 mgÆmL
)1
; Wako Pure Chemicals) was pre-
pared in methanol and stored at )80 °C until use. When
monensin or BFA was added to the medium, the methanol
concentration never exceeded 0.1%. Granulosa layer was
cultured at 41 °C in a humidified atmosphere of 5% CO
2
and 95% air. After culture, medium was collected and
stored at )20 °C. To separate the granulosacells and the
PL, the granulosa layer was placed into a drop of distilled
water (400 lL per piece) and washed with a flush of water
from a Pasteur pipette under a dissecting microscope.
Isolated PL was confirmed to be free from the granulosa
cells by phase contrast microscopy. After removal of the PL
and the BL, the residual solution, a mixture of intact
granulosa cells and cell debris, was confirmed not to contain
the PL and the BL by examination under a dissecting
microscope.
Electrophoresis and Western blot analysis
The PL and the suspension ofgranulosacells and cell debris
was solubilized in SDS/Tris (1% SDS buffered at pH 6.8
with 70 m
M
Tris/HCl). Insoluble materials were removed by
centrifugation at 14 500 g for 15 min and clear super-
natants served as PL lysates and total cell lysates. The
protein concentration in each sample was determined using
a BCA Protein Assay kit (Pierce, Rockford, IL, USA).
SDS/PAGE under nonreducing conditions was carried
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
)1
.
A peak at 6.1 min (48% acetonitrile) was collected, and the
N-terminal amino-acid sequence was confirmed as Ala318-
Arg-Asn-Thr-Trp-Val-Pro-Val-Glu-Gly327 by an auto-
mated gas-phase protein sequencer (Model 492, Applied
Biosystems).
Theexactmolecularmassofthisfragmentwasdeter-
mined using MALDI-TOF MS by means of a Voyager-DE
mass spectrometer (PE Biosystems) with a-cyano-4-
hydroxycinnamic acid (Aldrich Chemical) as a matrix.
In order to identify the C-terminal amino acid of ZPC,
18.5 lg of the purified ZPC as described above was applied
directly to an automated C-terminalprotein sequencer
(Procise 494-C, Applied Biosystems).
Production of antiserum against proZPC-derived peptide
A peptide (Pro-Val-Leu-Leu-Ser-Ala-Asp-Pro-Gly-Ala-
Val-Gly-Gln-Gln) corresponding to the sequence 376–389
of quailZPC coupled with an extra Cys residue at the N
terminus was synthesized using multiple peptide synthesizer
(SYRO II, MultiSynTech GmbH). A mature female rabbit
was immunized with the hemocyanin-coupled peptide
(200 l
4
g of peptide) as described previously [35].
N-Terminal sequence analysis of proZPC-derived peptide
Granulosa layers were cultured for 6 h in the presence of
200 ngÆmL
)1
monensin (1 mL medium per granulosa layer).
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
described previously [37] using anti-ZPC serum (1 : 300),
anti-(proZPC-derived peptide) serum (1 : 200), or normal
rabbit serum (1 : 200). The immunolabelled sections were
examined under an interference-contrast photomicroscope
(BX 50, Olympus Optics).
RESULTS
ZPC secretion by granulosa cells
Western blotting with anti-ZPC serum of the SDS-solubi-
lized granulosa cells, the PL, and the culture medium is
shown in Fig. 1. The lysates of the granulosacells before
culture were shown to contain three immunoreactive bands
of 35, 43, and 94 kDa (lane 1). The SDS-solubilized PL
contained only the 35-kDa protein (lane 2). After 6 h of
culture, a 35-kDa band was detected in the culture medium
(lane 5). The intensity of the 43-kDa band in the cell lysates
appeared to decrease during culture, whereas that of the
94-kDa band tended to increase (lane 3). From the
comparison of the intensity of the band as a proportion to
the total ZPCin the culture well, the amount of secreted
ZPC is larger than that of cellular ZPCafter culture for 6 h.
To refute the possibility of the release ofZPC from the PL in
the medium during culture, the isolated PL alone was
incubated for 6 h. Because the culture medium of the
isolated PL did not contain any immunoreactive bands
(lane 7), the 35-kDa immunoreactive proteinin the medium
must be secreted from the granulosacells during culture.
Next, we cultured granulosa layers for 8 h to assess the
time-related changes ofZPC contents in the medium and in
the cell lysates. As shown in Fig. 2A, the intensity of the
35-kDa band increased during culture. The content of
immunoreactive 43-kDa proteinin 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 secretionofZPC 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 ZPCsecretion (Fig. 4A). Although 25 ngÆmL
)1
BFA failed to affect the contents ofZPCin the cell lysates,
the addition of 50 ngÆmL
)1
BFA caused a distinct accumu-
lation of 43-kDa and 94-kDa proteins (Fig. 4B). The
addition of 100 ngÆmL
)1
BFA caused a decrease in the
35-kDa ZPC content of the cell lysates (Fig. 4B). These
results indicate that BFA inhibits the secretionofZPC by
inhibiting the conversion of proZPC to 35-kDa ZPC.
C-terminal sequence of 35-kDa ZPC
In order to determine the C-terminal amino acid of 35-kDa
ZPC, the electroblotted ZPC on PVDF membrane was
digested with lysylendopeptidase. As shown in Fig. 5A,
seven major bands were detected in the ZPC digests (lane 2).
From the amino-acid sequencing, we purified a 5.4-kDa
fragment by RP-HPLC. MALDI-TOF MS analysis
demonstrated that the molecular mass of this fragment is
Fig. 1. Western blot analysis ofZPCin medium, cells and PL. Gran-
ulosa layers were cultured for 0 (lanes 1 and 2) or 6 h (lane 3–5), and
the ZPCproteinin the granulosacells (lanes 1 and 3; 0.5 lgproteinper
lane; approximately 1/50 of the cell in one well), in the PL (lanes 2 and
4; 0.3 lg protein per lane; approximately 1/90 of the PL in one well)
and in the medium (lane 5, 8 lL culture medium per lane; 8/1000 of the
total volume in one well) were detected by using anti-ZPC serum
(1 : 2000 dilution). Isolated PL alone was also incubated for 6 h, and
theZPCproteininthePL(lane6;0.3lg protein per lane) and in the
medium (lane 7; 8 lL culture medium per lane) was analyzed.
Immunoblots shown are representative of at least three experiments.
Ó FEBS 2002 ProteolyticprocessingofZPCinquailgranulosa (Eur. J. Biochem. 269) 2225
Fig. 2. Time course ofZPC 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 proteinin 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 ZPCproteinin 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-terminalprotein sequencer.
Proteolytic processingof proZPC ingranulosa cells
In order to investigate the proteolyticprocessingof 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
ZPC but had only poor reactivity with 43-kDa ZPC (panels
1 and 3). This immunostaining was diminished by the
addition of antigen (Fig. 6, panel 4). These results indicate
that proZPC is cleaved between Phe360 and Pro376 during
proteolytic processing. In addition, anti-(proZPC-derived
peptide) serum detects the 12-kDa band (Fig. 6, panel 3),
which could not be detected by the anti-ZPC serum (Fig. 6,
panel 1). This suggests that the 12-kDa protein is the cleaved
peptide derived from the processingof proZPC.
Effects of monensin and BFA on proteolytic processing
of proZPC
Next, we evaluated the effects of monensin and BFA on the
proteolytic processingof proZPC. After the culture of the
granulosa layer with monensin or BFA, the cell lysates were
subjected to Western blot analysis using anti-(proZPC-
derived peptide) serum. As shown in Fig. 7A, the addition
of monensin caused an increase in the intensity of the
12-kDa band. This is consistent with the result shown in
Fig. 3B in which monensin inhibits the secretionof 35-kDa
ZPC but does not disturb the conversion of proZPC to
ZPC. On the other hand, > 80 ngÆmL
)1
BFA, which
inhibits the conversion of proZPC to ZPC (see Fig. 4B)
decreased the intensity of the 12-kDa band (Fig. 7B).
Detection of proZPC ingranulosa cells
To determine the localization of proZPC, the sections of
the granulosa layers were analysed by immunohistochem-
istry. As shown in Fig. 8A, the immunoreactive material
recognized by anti-ZPC serum was concentrated in the
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 ofZPC 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 ZPCin 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 ProteolyticprocessingofZPCinquailgranulosa (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
strong staining of the entire cytoplasm without swelling
(Fig. 8F). This staining pattern might reflect the accumu-
lation of proZPC in the granulosa cells.
N-Terminal sequence of the 12-kDa fragment derived
from proZPC
The 12-kDa fragment cleaved from proZPC was analysed
for the N-terminal amino-acid sequence. The first eight
residues are Asp-Ala-Gly-Lys-Glu-Val-Ala-Ala, which cor-
responds to the sequence Asp363–Ala370 deduced from the
cDNA. This result indicated that the proteolytic cleavage of
proZPC occurs at the consensus furin cleavage site, Arg359-
Phe360-Arg361-Arg362.
DISCUSSION
In the present study, we have shown that newly synthesized
proZPC is accumulated by inhibiting protein transport
from RER to the Golgi apparatus by BFA, and that ZPC
and the 12-kDa fragment generated by the proteolytic
processing of proZPC are accumulated by inhibiting protein
transport from the Golgi apparatus. As proZPC is not
secreted without proteolysis, this process might be a
prerequisite to ZPCsecretion and its incorporation into
the PL.
Fig. 7. Effects of monensin and BFA on pro-
teolytic processing. Granulosa layers were
cultured with monensin (0, 80, 160, 240, 320,
or 400 ngÆmL
)1
) or BFA (0, 20, 40, 60, 80, or
100 ngÆmL
)1
) 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 ZPCingranulosa layer. Sections ofgranulosa 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 ZPCsecretion without disturbing the conversion
of proZPC to ZPC (Fig. 3). On the other hand, monensin
impedes the proteolyticprocessingof pro-opiomelanocortin
in rat pituitary cells [42]. This might be due to the fact that
proteolytic processingof 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
cultured with BFA [45], the proteolytic conversion of
proalbumin to mature albumin takes place in the Golgi
apparatus [44]. Our findings regarding the accumulation of
proZPC in the presence of BFA and conversion of proZPC
to ZPCin the presence of monensin indicate that the
proteolytic processingofZPC could occur in the Golgi
apparatus.
Amino-acid sequence analysis showed that the C termi-
nus of mature ZPCprotein is Phe360 (Fig. 5). The
N-terminal amino acid of the proZPC-derived 12-kDa
fragment was determined to be Asp363, located just after
the consensus furin cleavage site. These results indicate that
the Arg361-Arg362 sequence might be missing. This may be
accounted for the following two possibilities: (a) proZPC is
initially digested at the consensus furin cleavage site and the
resulting C-terminal dibasic residues are trimmed off to
generate mature ZPC; and (b) proZPC directly receives the
proteolytic cleavage between Phe360 and Arg361, and the
N-terminal two residues of the proZPC-derived 12-kDa
fragment are trimmed off. In the case of neuropeptides and
peptide hormones, the C-terminal basic amino acid is
removed by a carboxypeptidase H in secretory granules
after initial digestion [46,47]. Although the N-terminal
amino-acid sequence of the proZPC-derived peptide was
not determined, Kubo et al. [48] reported that the two basic
C-terminal residues of gp43, a protein homologous to ZPC
in Xenopus laevis, is removed to produce the mature protein.
We think, therefore, that the processing event of proZPC to
ZPC inquailgranulosacells might take place initially by a
furin-like protease and then by a carboxypeptidase H-like
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 ofproteolyticprocessing 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 secretionof 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 proteinof 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 ofquail proZPC is considered to be, at least in
part, required for ZPCsecretion 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
selectively from the Golgi apparatus toward the apical
surface ofgranulosa cells, which are apposed to the PL. In
polarized Madin–Darby canine kidney cells, the O-glyco-
sylated domain has critical role for apical secretion of
neurotrophin receptors [52]. Fiedler et al. [53] reported that
antibody for annexin XIIIb significantly inhibited the
transport of influenza virus glycoprotein to the apical
plasma membrane. Efforts are currently in progress to
investigate the topology ofZPCsecretionin which ZPC
selectively secreted to the apical surface of the granulosa
cells forms the PL.
Our finding that the 12-kDa fragment cleaved from
proZPC accumulated in the monensin-treated cells
(Fig. 7A) and did not degrade immediately indicates the
possibility of the physiological importance of this fragment,
although its fate is currently unknown. The C-peptide, a
by-product ofproteolyticprocessingof proinsulin in the
pancreas, is demonstrated to have important physiological
effects on kidney and nerve functions, such that C-peptide
binds to specific G protein-coupled receptors on human
plasma membrane [54]. Nillni and Sevarino [23] also
described that the seven peptides derived from thyrotro-
pin-releasing hormone precursor are secreted from the
hypothalamus, and have various biological functions.
In the Western blot analysis, we found that the 94-kDa
band reacted with both anti-ZPC serum and anti-(proZPC-
derived peptide) serum in the cell lysates (Figs 1 and 6). This
protein migrates at the same position on SDS/PAGE under
reducing conditions as 43-kDa proZPC (data not shown).
The high molecular mass immunoreactive band was also
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 ZPCsecretion 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 ProteolyticprocessingofZPCinquailgranulosa (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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Ó FEBS 2002 ProteolyticprocessingofZPCinquailgranulosa (Eur. J. Biochem. 269) 2231
. determined as Phe by an
automated C-terminal protein sequencer.
Proteolytic processing of proZPC in granulosa cells
In order to investigate the proteolytic processing. Secretion of egg envelope protein ZPC after C-terminal proteolytic
processing in quail granulosa cells
Tomohiro Sasanami
1
, Jianzhi Pan
1
, Yukio