Báo cáo Y học: Secretion of egg envelope protein ZPC after C-terminal proteolytic processing in quail granulosa cells doc

Secretion of egg envelope protein ZPC after C-terminal proteolytic processing in quail granulosa cells Tomohiro Sasanami1, Jianzhi Pan1, Yukio Doi2, Miki Hisada3, Tetsuya Kohsaka1, Masar

Secretion of egg envelope protein ZPC after C-terminal proteolytic processing in quail granulosa cells

Tomohiro Sasanami1, Jianzhi Pan1, Yukio Doi2, Miki Hisada3, Tetsuya Kohsaka1, Masaru Toriyama1 and Makoto Mori1

1

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Japan;2Department of Food Science, Kyoto Women’s University, Higashiyama, Kyoto, Japan;3Suntory 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 ZPCand the secretion of ZPCfrom 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 proZPCin the

pres-ence of brefeldin A, and conversion of proZPCto ZPCin

the presence of monensin, indicate the possibility that the

proteolytic processing of ZPCoccurs 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 ZPCis cleaved at the consensus furin cleavage site, and the resulting two basic residues at the Cterminus are subsequently trimmed off to generate mature ZPCprior 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 egg envelope [2–6]

The egg envelope 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 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

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 ZPCfrom the comparison

of deduced amino-acid sequences of the known ZPC Avian ZPCwas 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 ZPCproduction provides a beneficial model for study of the vectorial

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 (EC3.4.21.50).

Note: the GenBank accession number of proteins mentioned in the text

are: 33-kDa protein in quail (quail ZPC), AB012606; 32-kDa protein

in chicken (chicken ZPC), D89097.

(Received 23 October 2001, revised 26 February 2002,

accepted 12 March 2002)

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 secretion of the

mature ZPCfrom 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]

M A T E R I A L S A N D M E T H O D S

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 mM; Wako Pure Chemicals)

and BFA (5 mgÆmL)1; Wako Pure Chemicals) was

pre-pared in methanol and stored at)80 Cuntil use When

monensin or BFA was added to the medium, the methanol

concentration never exceeded 0.1% Granulosa layer was

cultured at 41Cin a humidified atmosphere of 5% CO2

and 95% air After culture, medium was collected and

stored at)20 C To separate the granulosa cells 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 of granulosa cells and cell debris

was solubilized in SDS/Tris (1% SDS buffered at pH 6.8

with 70 mMTris/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

Durham, NC, USA) as a secondary antibody

Determination of the C-terminus of ZPC ZPCwas 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 ZPCwas digested at 37Cfor 16 h with 400 pmol lysylendopepti-dase (EC3.4.21.50, Wako Pure Chemicals) dissolved in 10% acetonitrile buffered at pH 9.0 with 50 mMTris/HCl The ZPCdigests 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)

The exact molecular mass of this fragment was deter-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 ZPCas described above was applied directly to an automated C-terminal protein 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 quail ZPCcoupled 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)1monensin (1 mL medium per granulosa layer) After culturing, the granulosa layer was extracted with ice-cold RIPA buffer (300 mMNaCl, 2% Nonidet P-40, 1% deoxycholate, 0.2% SDS, 50 mMTris/HCl pH 7.5) at 4C for 16 h Insoluble constituents were removed by centrifu-gation at 14 500 g at 4Cfor 20 min and the supernatant served as granulosa cell extracts

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 4C 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 proZPCand 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-ZPCserum (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)

R E S U L T S

ZPC secretion by granulosa cells

Western blotting with anti-ZPCserum of the

SDS-solubi-lized granulosa cells, the PL, and the culture medium is

shown in Fig 1 The lysates of the granulosa cells 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 ZPCis larger than that of cellular ZPCafter culture for 6 h

To refute the possibility of the release of ZPCfrom 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 protein in the medium must be secreted from the granulosa cells during culture Next, we cultured granulosa layers for 8 h to assess the time-related changes of ZPCcontents 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 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 ZPCwas 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 ZPCsecretion In contrast, an increase in the intensity of all of the bands in the cell lysates was observed with the addition of 160 ngÆmL)1of monensin (Fig 3B) Thus, monensin inhibits the secretion of ZPCwithout interfering with the conversion of proZPCto 35-kDa ZPC Effect of BFA on ZPC secretion

We next investigated the effects of BFA on ZPCsecretion The addition of 50 ngÆmL)1BFA caused a decrease in ZPC contents in the medium, and 100 ngÆmL)1BFA completely abolished ZPCsecretion (Fig 4A) Although 25 ngÆmL)1 BFA failed to affect the contents of ZPCin the cell lysates, the addition of 50 ngÆmL)1BFA 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 ZPCcontent of the cell lysates (Fig 4B) These results indicate that BFA inhibits the secretion of ZPCby inhibiting the conversion of proZPCto 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 ZPCdigests (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 of ZPC in medium, cells and PL

Gran-ulosa layers were cultured for 0 (lanes 1 and 2) or 6 h (lane 3–5), and

the ZPCprotein in the granulosa cells (lanes 1 and 3; 0.5 lg protein per

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-ZPCserum

(1 : 2000 dilution) Isolated PL alone was also incubated for 6 h, and

the ZPC protein in the PL (lane 6; 0.3 lg 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.

Fig 2 Time course of ZPC content in the medium and the cell lysate during 8h of culture Granulosa layers were cultured for 0, 2, 4, 6, or 8 h, and ZPCprotein in the medium (A) and in the cell lysate (B) were detected by using anti-ZPCserum 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)1monensin for 6 h The ZPC protein in the medium (A) and in the cell lysate (B) were detected by using anti-ZPCserum 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)1BFA for 6 h The ZPCprotein in the medium (A) and in the cell lysate (B) were detected by using anti-ZPCserum Values are means ± SEM of three independent experiments with triplicate wells.

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-ZPCserum, anti-(proZPC

-derived peptide) serum tended to react well with the 94-kDa

ZPCbut 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 proZPCis 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-ZPCserum (Fig 6, panel 1) This suggests that the 12-kDa protein is the cleaved peptide derived from the processing of proZPC

Effects of monensin and BFA on proteolytic processing

of proZPC Next, we evaluated the effects of monensin and BFA on the proteolytic processing of 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 secretion of 35-kDa ZPCbut does not disturb the conversion of proZPCto ZPC On the other hand, > 80 ngÆmL)1 BFA, which inhibits the conversion of proZPCto ZPC(see Fig 4B) decreased the intensity of the 12-kDa band (Fig 7B) Detection of proZPC in granulosa 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-ZPCserum 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 proZPCin 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 ZPCdigested 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-ZPCserum (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).

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 proZPCin the granulosa cells

N-Terminal sequence of the 12-kDa fragment derived

from proZPC

The 12-kDa fragment cleaved from proZPCwas 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 proZPCoccurs at the consensus furin cleavage site, Arg359-Phe360-Arg361-Arg362

D I S C U S S I O N

In the present study, we have shown that newly synthesized proZPCis 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 proZPCare accumulated by inhibiting protein transport from the Golgi apparatus As proZPCis 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 proZPCprotein in the cell lysate (0.5 lg protein per lane) 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)1monensin (E) and with 100 ngÆmL)1BFA (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.

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 proZPCto 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

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

proZPCin the presence of BFA and conversion of proZPC

to ZPCin the presence of monensin indicate that the

proteolytic processing of ZPCcould occur in the Golgi

apparatus

Amino-acid sequence analysis showed that the

Ctermi-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) proZPCis

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 proZPCto

ZPCin quail granulosa cells might take place initially by a

furin-like protease and then by a carboxypeptidase H-like

protease On the other hand, mouse ZPCwas 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 ZPCbiosynthesis

Our results demonstrated that ZPCis 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 ZPCfrom 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 proZPCis considered to be, at least in

part, required for ZPCsecretion rather than ZPCbiosyn-thesis The consensus furin cleavage site was found within the hydrophobic domain near the Cterminus in the ZPCof 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 ZPCfrom 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 of granulosa 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 of ZPCsecretion in which ZPC selectively secreted to the apical surface of the granulosa cells forms the PL

Our finding that the 12-kDa fragment cleaved from proZPCaccumulated 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 of proteolytic processing of 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-ZPCserum 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 43-94-kDa proZPCgenerated during post-translational modification

In conclusion, our study suggests that newly synthesized ZPCis 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 ZPCprior to secretion This process might be a prerequisite event for ZPCsecretion and its incorporation into PL

A C K N O W L E D G E M E N T S

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

grant-in-aid for scientific research (09660300, 11660280, and 13660284

to M M.) from the Ministry of Education, Science, Sports, and

Culture, Japan.

R E F E R E N C E S

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