Localization of N-linked carbohydrate chains in glycoprotein ZPA
of the bovine egg zona pellucida
Keiichi Ikeda
1
, Naoto Yonezawa
1,2
, Keita Naoi
1
, Toshiyuki Katsumata
3
, Seizo Hamano
4
and
Minoru Nakano
1,2
1
Graduate School of Science and Technology and
2
Department of Chemistry, Chiba University, Japan;
3
College of Liberal Arts
and Science, Tokyo Medical and Dental University, Chiba, Japan;
4
Animal Bio-Technology Center,
Livestock Improvement Association, Tokyo, Japan
The zona pellucida, a transparent envelope surrounding the
mammalian oocyte, consists of three glycoproteins, ZPA,
ZPB and ZPC, and plays a role in sperm–egg interactions. In
bovines, these glycoproteins cannot be separated unless the
acidic N-acetyllactosamine regions of the carbohydrate
chains are removed by endo-b-Galactosidase digestion.

The mammalian oocyte is coated with a transparent matrix
called the zona pellucida. This matrix plays various roles in
the early phase of fertilization: species-specific sperm
binding, blocking polyspermy, and protecting the embryo
until implantation [1,2]. The zona is composed of three
glycoproteins, called ZPA, ZPB and ZPC in the order of the
size of their cDNAs [3], and their carbohydrate chains are
responsible for species-specific sperm-zona binding [1,4].
In the pig, the carbohydrate structures of O-linked chains
of zona protein mixture [5,6] and the acidic and neutral
N-linked chains of ZPB/ZPC mixture [7,8] are well charac-
terized. Sperm-binding activity has been ascribed to the
O-linked chains of the zona protein mixture [9] or to the
neutral N-linked chains obtained from the ZPB/ZPC
mixture [8]. We have shown that the triantennary/tetraan-
tennary chains of the ZPB/ZPC mixture possess greater
activity than the diantennary chains [10]. The triantennary/
tetraantennary chains of ZPB are localized at Asn220 in the
N-terminal region [10]. Moreover, the isolated N-terminal
peptide of ZPB including Asn220 has sperm-binding activity
[11]. Yurewicz et al. [12] showed that ZPB and ZPC form
heterocomplexes that have sperm-binding activity, but that
monomeric ZPB or ZPC does not exhibit this activity.
These results suggest that ZPC contributes to the expression
of the sperm-binding activity of the neutral N-linked chain
of ZPB. In ZPC, the triantennary/tetraantennary chains are
mainly localized at Asn271 in the C-terminal region [13].
The sperm-binding activity of porcine ZPA, a minor
component, has not been assessed because its purification
is difficult.

trypsin (EC 3.4.21.4); N-glycanase (EC 3.5.1.52)
(Received 22 April 2002, revised 21 June 2002, accepted 12 July 2002)
Eur. J. Biochem. 269, 4257–4266 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03111.x
ZPC has weak activity. However, it is unclear whether ZPA
possesses sperm-binding activity using in vitro competition
and sperm-bead binding assays [18]. We deduced the amino
acid sequence of the bovine ZPA by cDNA cloning and
sequencing, and revealed that bovine ZPA has four
potential N-glycosylation sites [18], whereas porcine ZPA
has six potential sites [3]. Here, we describe the localization
of the N-linked chains in bovine ZPA.
MATERIALS AND METHODS
Preparation of zona protein mixture
Zonae pellucidae of bovine eggs were isolated from frozen
ovaries as described previously [16] and were solubilized in
H
2
Oat70°C for 30 min and lyophilized. The yield of
bovine zona proteins from one ovary is approximately one-
tenth of that of porcine zona proteins. Total 1.2 mg of
protein mixture (60 000 zonae) from 2400 ovaries were used
for the present experiment. The heat solubilized zonae were
digested with Escherichia freundii endo-b-Galactosidase
(Seikagaku co., Tokyo, Japan) in 0.5
M
ammonium acetate
(pH 5.6) at 37 °C for 48 h [8].
Tryptic digestion and cyanogen bromide cleavage
of zona protein mixture
After the endo-b-Galactosidase-digested zona protein mix-

After elution with 21 mL of buffer A, the materials retained
in the column were eluted with 0.2
M
methyl-a-
D
-manno-
pyranoside/buffer A. The flow-through fraction was applied
to a Canavalia ensiformis agglutinin (ConA) agarose column
(13 · 23 mm; Seikagaku co.). Elution conditions were the
same as those in the case of the LCA column.
Fractionation of N-glycopeptides
The LCA-binding fraction and the ConA-binding fraction
were separately applied to a Chemcosorb 3C
8
HPLC
column (4.6 · 150 mm; Chemco, Osaka, Japan) equili-
brated with 0.1% trifluoroacetic acid and eluted by a linear
gradient from 0 to 50% acetonitrile in 0.1% trifluoroacetic
acid over 50 min at a flow rate of 1 mLÆmin
)1
at 37 °C.
Effluent was monitored at 230 nm. The N-terminal amino
acid sequence of each peak was determined by automated
Edman degradation using a PPSQ-21 protein sequencer
(Shimadzu, Kyoto, Japan).
Purification of ZPA
The endo-b-Galactosidase-digested zona protein mixture
was fractionated into three components (ZPA–C) on the
Nucleosil 300–7C
18

described above. Tryptic digests were applied to a Chem-
cosorb 3C
8
column. Because elution conditions were the
same as in the case of the separation of N-glycopeptides
from the zona protein mixture (Fig. 1), the elution times of
the tryptic N-glycopeptides from ZPA were the same as
those shown in Fig. 1A and B. Each fraction containing
N-glycopeptide was digested with N-glycanase (0.5 mU)
in 0.1
M
Tris/HCl (pH 8.6) at 37 °C for 18 h. The digests
were applied to tandem-linked columns of AG50W-X8
(10 · 15 mm; Bio-Rad, California, USA) and AG3
(10 · 15 mm, Bio-Rad), and the columns were eluted with
water. The flow-through fraction was collected. After
lyophilization, the N-linked chains were then digested with
Jack bean b-Galactosidase (Seikagaku co.) in 50 lLof
0.1
M
sodium citrate (pH 4.1) at 37 °C for 24 h. After the
pH of the digestion mixture was adjusted to 8.0 by 1
M
Tris/
HCl (pH 8.6), the solutions were applied to the AG3
column. The flow-through fraction with water was collected
and lyophilized.
Pyridylamination of N-linked oligosaccharides
The N-linked oligosaccharides thus obtained were modified
with 2-aminopyridine and sodium cyanoborohydride [20].

)1
. The fluorescence intensity was monitored
with excitation at 310 nm and emission at 375 nm.
Sugar mapping analysis of the pyridylaminated
N-linked chains
The pyridylaminated neutral fraction of N-linked chains
was chromatographed on a Shim-pack CLC-ODS column
(6 · 150 mm; Shimadzu) by a linear gradient from 0.1 to
0.25% 1-butanol in 10 m
M
sodium phosphate (pH 3.8) for
60 min at a flow rate of 1 mLÆmin
)1
at 55 °C[14].The
fluorescence intensity was monitored with excitation at
320 nm and emission at 400 nm. Major peaks were further
chromatographed on a size-fractionation HPLC column of
TSK gel Amide-80 (4.6 · 250 mm; Tosoh). Elution was
performed by a linear gradient decrease in acetonitrile from
65 to 50% in 0.5
M
acetic acid/triethylamine (pH 7.3) over
60 min at a flow rate of 1 mLÆmin
)1
at 40 °C. The
fluorescence intensity was monitored with excitation at
310 nm and emission at 375 nm. The pyridylaminated
acidic fractions were also analyzed using both columns after
digestion with Arthrobacter ureafaciens sialidase (Nacalai
tesque, Kyoto, Japan) in 0.1

were then fractionated by reverse-phase HPLC separately.
N-terminal amino acid sequence analysis of each peak of the
tryptic peptides revealed that the peptides in the three peaks
in Fig. 1A and B (peak a at 19 min, peak b at 34 min and
peakcat38min)arefragmentsofZPA.Thatis,
Fig. 1. Reverse-phase HPLC of N-glycopep-
tides retained to LCA and ConA agarose.
Reduced and carboxymethylated zona protein
mixture was cleaved by trypsin and BrCN and
appliedtoanLCAagarosecolumn.Theflow-
throughfractionfromLCAcolumnwas
applied to a ConA agarose column. Fractions
eluted with 0.2
M
methyl-a-
D
-mannopyrano-
side from both columns were subjected to
Chemcosorb 3C
8
HPLC. Elution was per-
formed with a linear gradient of acetonitrile
(broken line) in 0.1% trifluoroacetic acid.
(A and B) Tryptic peptides; (C and D) BrCN-
peptides; (A and C) LCA binding fraction;
(B and D) ConA binding fraction. Peptides
were detected at 230 nm. Arrowheads indicate
the elution positions of the N-glycopeptides
from ZPA.
Ó FEBS 2002 Localization of N-linked chains in ZPA (Eur. J. Biochem. 269) 4259

Asn527.
Sugar mapping of the carbohydrate chains from each
N-glycosylation site
Three tryptic N-glycopeptides from the endo-b-Galactos-
idase-digested ZPA were eluted at the same positions as
peaks a–c in Fig. 1. N-linked chains were obtained by
N-glycanase digestion of these tryptic peptides. We iden-
tified the structures of the N-linked chains of ZPA by two-
dimensional mapping on HPLC referring to the reported
structures of the unfractionated zona protein mixture [14].
To obtain the core carbohydrate chains of the acidic
chains, the N-linked chains were further digested with
b-Galactosidase [14]. After pyridylamination, the N-linked
chains were separated into neutral and acidic fractions by
DEAE-5PW HPLC (Fig. 4). The elution position of the
acidic fraction was the same as that of the monosialylated
chain, and all the acidic chains in this fraction were
neutralized by sialidase digestion. From the peak area, the
molar ratios of the neutral chains to the acidic chains
linked to Asn83, Asn191 and Asn527 were estimated to be
78 : 22, 67 : 33 and 67 : 33, respectively. The neutral
chains were subjected to two-dimensional sugar mapping
and the acidic chains were also analyzed by mapping after
digestion with sialidase. The glucose units in the mapping
are summarized in Table 1. The high-mannose-type chain,
Fig. 3. N-glycosylation sites of bovine ZPA. The amino acid sequence
of bovine ZPA, as deduced from cDNA and N-terminal amino acid
sequence analyses [16,18]. Solid and dotted underlines indicate the
N-terminal amino acid sequences of BrCN and tryptic N-glycopep-
tides, respectively. Filled black boxes indicate the N-glycosylation sites

type chains, as these sialylated b-Gal residues were
unsusceptible to b-Galactosidase. In a previous experiment
[14], sugar mapping analysis was applied to the N-linked
chains from the zona protein mixture after digestion with
sialidase, endo-b-Galactosidase and b-Galactosidase.
Therefore, the nonreducing terminal b-Gal residues in the
sialidase digests of the acidic fraction in Table 1 have been
eliminated and the monosialylated chains converge on the
chains in the neutral fraction.
DISCUSSION
The molar ratios of ZPA/ZPB/ZPC in the porcine and
bovine zona pellucida are 1 : 3 : 3 and 1 : 1 : 2, respectively
[17,23]. Recently, the neutral N-linked chains in porcine
ZPB and ZPC were localized [10,13], but no information on
the structures of these carbohydrate chains or their local-
ization in the minor component porcine ZPA is available,
because its purification is difficult. In this study, we localized
a neutral high-mannose-type chain and acidic complex-type
chains in bovine ZPA in order to fill a gap in the structural
information on the zona glycoproteins. As with porcine
ZPB and ZPC, bovine ZPA possesses three N-glycosylation
sites (Fig. 2) determined to be Asn83, Asn191 and Asn527
in amino acid sequence analyses of tryptic and BrCN
peptides.
The acidic property of the N-linked chains of bovine zona
proteins is due to sialic acids [14], whereas many sulfates are
linked to the N-acetyllactosamine repeats of porcine zona
proteins [7,24]. The N-linked chains of the bovine zona
Fig. 4. Anion-exchange HPLC of N-linked chains from tryptic N-gly-
copeptides. After b-Galactosidase digestion, N-linked chains of each

binding has not been demonstrated. Bovine sperm-binding
activity is mainly ascribed to the high-mannose-type chain
with five mannose residues [15].
The ZP domain is a module, approximately 260 resi-
dues long, common to all three zona protein components
[25–27] and this domain contains eight conserved cysteine
residues, which form four disulfide linkages [25]. Bovine
ZPA has only one N-glycosylation site in the ZP domain,
while porcine ZPB and ZPC have three N-glycosylation
Table 1. Glucose units of the N-linked chains from each of the N-glycosylation sites of bovine ZPA. The values in parentheses are glucose units of
authentic pyridylaminated carbohydrate chains [14]. Molar ratio of the N-linked chains were calculated from the peak area. Proposed structures are
from references [14,21]. PA, pyridylamino.
Number of glucose unit
Site
N-linked
chain
Shim-pack
CLC-ODS Amide 80 Structure Molar ratio
4262 K. Ikeda et al.(Eur. J. Biochem. 269) Ó FEBS 2002
sites in the domain (Fig. 5). Furthermore, porcine ZPA
has one potential N-glycosylation site in the domain,
Asn530. Therefore, the N-glycosylation sites in the ZP
domain are not conserved in the three zona protein
components. In the pig, triantennary and tetraantennary
neutral complex-type chains have sperm-binding activity
[10] and these active chains are mainly localized in the
N-terminal region of the ZP domain of ZPB and the
C-terminal region of the ZP domain of ZPC (Fig. 5). In
the cow, ZPB exhibits the strongest sperm-binding
activity among the components and ZPC exhibits

little of it at the N-glycosylation site, Asn527, in the ZP
domain. This may explain why the sperm-binding activity
of ZPA is weak, if any. The localization of the sperm-
ligand active high-mannose-type chain in bovine ZPB and
ZPC should be clarified.
Besides the zona proteins, the ZP domain has been found
in several proteins: TGF-b type III receptor (betaglycan)
from fetal and adult tissues [28,29], uromodulin (urinary
Tamm-Horsfall glycoprotein of pregnant woman) [30], the
major zymogen granule membrane protein (GP-2) [31], and
the proteins in the uterus and oviduct [32,33]. Furthermore,
tectorins in the tectorial membranes responsible for hearing
also have a ZP domain [34,35]. In each protein, the ZP
domain is generally present next to a putative transmem-
brane region [25]. The structural characterization of the
carbohydrate chains in the ZP domain of these proteins is of
interest.
ACKNOWLEDGMENT
This study was supported in part by a Grant-in Aid for Scientific
Research from the Ministry of Education, Science and Culture of
Japan.
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