Tài liệu Báo cáo khoa học: Localization of N-linked carbohydrate chains in glycoprotein ZPA of the bovine egg zona pellucida pptx

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.

Endo-b-Galactosidase-digested ZPB retains stronger sperm-
binding activity than ZPC. It is still unclear whether ZPA
possesses significant activity. Recently, we reported that
bovine sperm binds to Man
5
GlcNAc
2
, the neutral N-linked
chain in the cow zona proteins. In this study, we investigated
the localization of the sperm-ligand active high-mannose-
type chain and the acidic complex-type chains in bovine
ZPA. Three N-glycopeptides of ZPA, containing an N-gly-
cosylation site at Asn83, Asn191 and Asn527, respectively,
were obtained from endo-b-Galactosidase-digested ZPA. Of
these glycosylation sites, only Asn527 is present in the ZP
domain common to all the zona proteins. The carbohydrate
structures of the N-linked chains obtained from each
N-glycopeptide were characterized by two-dimensional
sugar mapping analysis, while considering the structures of
the N-linked chains of the zona protein mixture reported
previously. Acidic complex-type chains were found at all
three N-glycosylation sites, while Man
5
GlcNAc
2
was found
at Asn83 and Asn191, but there was very little of this sperm-
ligand active chain at Asn527 in the ZP domain of ZPA.
Keywords: glycoprotein; N-linked carbohydrate chain;
sperm ligand; zona pellucida; ZP domain.

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.

The N-linked chains of the bovine zona protein mixture
include neutral (23%) and acidic (77%) chains [14]. We
have determined the structures of the neutral chain and the
core regions of the acidic chains [14]. The acidic chains are
diantennary, triantennary and tetraantennary, fucosylated
complex-type chains with a tandem N-acetyllactosamine
repeat in the nonreducing regions to which the main sialic
acids are attached. The neutral chain is a high-mannose-
type chain with five mannose residues (Man
5
GlcNAc
2
).
This high-mannose-type chain exhibits sperm-binding
activity and inhibits in vitro fertilization [15].
The native bovine zona proteins cannot be separated into
their three components because their molecular masses are
almost identical (74 kDa) and their carbohydrate chains are
very heterogeneous [16]. When they are digested with endo-
b-Galactosidase to remove the acidic N-acetyllactosamine
repeat, the three components become separable [16–18].
Sperm-binding activity is mainly ascribed to ZPB, while
Correspondence to M. Nakano, Department of Chemistry,
Faculty of Science, Chiba University, 1-33, Yayoi-cho,
Inage-ku, Chiba-shi, Chiba, Japan 263-8522.
Fax: + 81 43 2902874, E-mail:
Abbreviations: BrCN, cyanogen bromide; LCA, Lens culinaris
agglutinin; ConA, Canavalia ensiformis agglutinin.
Enzymes: b-Galactosidase (EC 3.2.1.23); keratan-sulfate
endo-1,4-b-Galactosidase (EC 3.2.1.103); sialidase (EC 3.2.1.18);

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-

ture was reduced and carboxymethylated as described
previously [13], the protein mixture (400 lg) was digested
with trypsin (enzyme/substrate 1 : 100, w/w) in NaCl/P
i
(pH8.0)at37°C for 18 h. The protein mixture (400 lg)
was also cleaved by cyanogen bromide (BrCN) in 70%
formic acid at room temperature for 18 h in the dark and
the products were desalted by using a Nucleosil 300–7C
18
column (4 · 150 mm; Machery-Nagel, Duren, Germany).
Elution was performed by a gradient of acetonitrile from 0
to 50% in 0.1% trifluoroacetic acid at a flow rate of
1mLÆmin
)1
at 37 °C.
Lectin affinity chromatography of glycopeptides
Tryptic peptides and BrCN-peptides dissolved in 500 lL
of buffer A (1 m
M
calcium chloride, 1 m
M
magnesium
chloride, 150 m
M
sodium chloride, 0.02% sodium azide,
10 m
M
Tris/HCl, pH 7.2) were separately applied to
a Lens culinaris agglutinin (LCA) agarose column
(13 · 23 mm; Seikagaku co.) equilibrated with buffer A.

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

column [18] at 40 °C.
N-glycanase digestion of ZPA
We showed that ZPA is specifically cleaved between Ala167
and Asp168 by an unknown protease on fertilization, giving
a short N-terminal fragment (36–167) and a long C-terminal
fragment (168–637), which are crosslinked via a disulfide
bond [16,17]. This site is also cleaved in at least half of the
ZPA molecules during preparation of the zona from
ovarian eggs. The molecular masses of the N- and
C-terminal fragments of the endo-b-Galactosidase-digested
ZPA are 21 and 63 kDa, respectively [12], and the time
course of the N-glycanase (glycopeptidase F; Takara Shuzo,
Kyoto, Japan) digestion of these fragments could be
monitored by SDS/PAGE on 15 and 8% acrylamide gels,
respectively, under reducing conditions [17]. The digestion
was performed in 0.1% SDS, 10 m
M
o-phenanthroline,
100 m
M
sodium phosphate (pH 8.6) at 37 °C and aliquots
were removed at various times between 0 min and 22 h and
subjected to SDS/PAGE. To detect the shift of the 21 kDa
band, 2.5 mU of N-glycanase was added to 4 lg (56 pmol)
of ZPA. To detect the shift of the 63 kDa band, 0.25 mU of
N-glycanase was added to 1 lg (14 pmol) of ZPA.
Isolation of core regions of N-linked oligosaccharides
from glycopeptides
After reduction and carboxymethylation [13], the purified
ZPA (21 lg) was digested with trypsin under the conditions

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].

4258 K. Ikeda et al.(Eur. J. Biochem. 269) Ó FEBS 2002
The excess reagents were removed by phenol/chloroform
(1 : 1, v/v) extraction performed three times. The aqueous
phase was applied to a Cellulofine GCL25-sf HPLC column
(7.5 · 600mm;Seikagakuco.),andelutedwith10%
acetonitrile/10 m
M
ammonium acetate at a flow rate of
0.3 mLÆmin
)1
. The fluorescence intensity of the effluent was
monitored with excitation at 320 nm and emission at
400 nm.
Separation of pyridylaminated neutral and acidic chains
The pyridylaminated N-linked oligosaccharides were sepa-
rated into neutral and acidic fractions by using a TSK-gel
DEAE-5PW HPLC column (7.5 · 75 mm; Tosoh, Tokyo,
Japan) equilibrated with NH
3
/H
2
O (pH 9.0). Elution was
performed by a linear gradient from 0 to 100% of 0.5
M
CH
3
COONH
4
(pH 8.0) over 60 min at a flow rate of
0.5 mLÆmin

)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

M
ammonium acetate (pH 5.0)
at 37 °C for 24 h. The glucose units of the pyridylaminated
N-linked chains were calculated from their retention times
using the pyridylaminated glucose oligomers as the standard
[14,21].
RESULTS
Identification of N-glycosylation sites
The acidic chains of the bovine zona protein mixture are
diantennary, triantennary and tetraantennary, sialylated
complex-type chains with a fucose residue at their reducing
ends, while the neutral chain is a high-mannose-type chain
[14]. The endo-b-Galactosidase-digested zona protein mix-
ture was cleaved using trypsin and BrCN. The tryptic
peptides and BrCN-peptides were separately applied to an
LCA agarose column, which binds to the Fuc(a1–6)Glc-
NAc of the complex-type chains [22]. The glycopeptides
bound to the LCA column were eluted with 0.2
M
a-methyl-
D
-mannopyranoside. The flow-through fraction from the
LCA column was applied to a ConA agarose column,
which binds to the high-mannose-type chain. The glyco-
peptides retained in the column were eluted with 0.2
M
a-methyl-
D
-mannopyranoside. The LCA-binding fraction
(Fig. 1A,C) and the ConA-binding fraction (Fig. 1B,D)

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

VLXRTDPNIK (peak a), MLXCTYVLDP (peak b) and
AQXLTLQEALTQGYNLLIEN (peak c) correspond to
the N-terminal sequences of tryptic peptides 525–534, 81–92
and 189–210, respectively, in which X is a glycosylated Asn
residue (Fig. 3). Furthermore, the three peaks in Fig. 1C
(peak 1 at 34 min, peak 2 at 38 min and peak 3 at 40 min)
were shown to be fragments of ZPA. That is,
LXCTYVLDPE (peak 1), GWTVTVGDGE (peak 2)
and LINTNVESLP (peak 3) correspond to the N-terminal
sequence of BrCN-peptides 82–112, 178–211 and 468–546,
respectively (Fig. 3). The absence of peak 3 in Fig. 1D
agrees with the fact that there is only a small amount of the
high-mannose-type chain at Asn527 (see below). Collec-
tively, Asn83, Asn191 and Asn527 of ZPA are glycosylated.
Determination of the number of N-glycosylation sites
We then investigated the number of N-glycosylation sites
and the localization of N-linked chains in ZPA using the
purified ZPA. In the 15% gel, the 21 kDa band shifted to
16 kDa within 2 min and no further time-dependent shift
was observed within 22 h (Fig. 2A), indicating that one
N-glycosylation site is present in the N-terminal fragment.
This fragment has a potential N-glycosylation site at Asn83
(Fig. 3). In the 8% gel, the 63 kDa band shifted to 59 and
56 kDa bands within five minutes, but the 59 kDa band did
not converge on the 56 kDa within 22 h (Fig. 2B). The
carbohydrate chains linked to one of the two sites may be
resistant to N-glycanase, although the reason is uncertain.
These results confirmed that two N-glycosylation sites are
present in the C-terminal fragment and that the
N-glycosylation sites of ZPA are Asn83, Asn191 and

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

determined by the present experiment and the broken box indicates a
potential N-glycosylation site that is not glycosylated. The arrow
indicates the N-terminus of mature ZPA and arrowhead indicates the
specific cleavage site on fertilization. The putative processing site for
furin or furin-like enzymes is double underlined. Closed area of
Leu366-Ser632 indicates ZP domain [18,25] and the putative trans-
membrane domain is from Thr683 to Leu697.
Fig. 2. Time course of N-glycanase digestion of ZPA. At least half of
the ZPA molecules are specifically cleaved by an unknown protease
during preparation, giving rise to N-terminal 21 kDa fragment and
C-terminal 63 kDa fragment, which are crosslinked through a disulfide
bond. This ZPA specimen was digested with N-glycanase at 37 °C. At
various times, aliquots were removed and subjected to SDS/PAGE on
a 15% for the 21 kDa fragment (A) and 8% for the 63 kDa fragment
(B) separating gel under reducing conditions. Proteins were silver-
stained. The bands moving faster on the SDS gel than the band of
undigested ZPA fragments (0 min) are indicated by arrowheads.
4260 K. Ikeda et al.(Eur. J. Biochem. 269) Ó FEBS 2002
which is the intact neutral chain [14], was found at Asn83
and Asn191, but was rare at Asn527. The other chains in
the neutral fraction in Table 1 must be neutralized by
endo-b-Galactosidase digestion, as their sialic acids are
originally linked to the N-acetyllactosamine repeats. While
all the chains in the neutral fraction were found previously
by analysis of the unfractionated zona protein mixture [14],
the monosialylated complex-type chains in the acidic
fraction on DEAE-HPLC (sialidase digests of the acidic
fraction in Table 1) were found in this analysis of ZPA.
These sialic acids must be linked to the nonreducing
terminal b-Gal residues of the core regions of the complex-

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

tryptic N-glycopeptide from endo-b-Galactosidase digested ZPA were
pyridylaminated and applied to a DEAE-5PW HPLC column equili-
brated with NH
3
/H
2
O (pH 9). Elution was performed by a linear
gradient and from 0 to 100% of 0.5
M
CH
3
COONH
4
(pH 8) for
60 min at a flow rate of 0.5 mLÆmin
)1
. The fluorescence intensity was
monitored with excitation at 310 nm and emission at 375 nm. Arrow
heads and arrows indicate the elution positions of neutral chains and
monosialylated acidic chains, respectively. (A) peak a (Asn527); (B)
peak b (Asn83) and (C) peak c (Asn191).
Fig. 5. N-glycosylation sites in ZP domain among ZP components.
N-glycosylation sites of bovine ZPA and porcine ZPB [10,11] and ZPC
[13] in the ZP domains are shown by filled black boxes. Arrows and C
indicate the linkage sites of sperm-ligand carbohydrate chains and
positions of cysteine residues, respectively.
Ó FEBS 2002 Localization of N-linked chains in ZPA (Eur. J. Biochem. 269) 4261
proteins possess one to several sialic acid residues and most
of these are eliminated on fertilization [14]. However,
participation of the sialic acids in bovine sperm-zona

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

sperm-binding activity approximately one-sixth that of
ZPB [18]. However, it is still unclear whether bovine ZPA
plays a role in sperm-zona binding. That is, ZPA exhibits
weak sperm-binding activity in the competition assay, but
does not exhibit activity in the sperm-bead binding assay
[18]. Table 1 shows that bovine ZPA possesses the sperm-
ligand active high-mannose-type chain, but there is very
Table 1. (Continued).
Number of glucose unit
Site
N-linked
chain
Shim-pack
CLC-ODS Amide 80 Structure Molar ratio
Ó FEBS 2002 Localization of N-linked chains in ZPA (Eur. J. Biochem. 269) 4263
Table 1. (Continued).
Number of glucose unit
Site
N-linked
chain
Shim-pack
CLC-ODS Amide 80 Structure Molar ratio
In 83A1, 191A1 and 527A1, nonreducing terminal b-Gal residue attaches to GlcNAc residue of the a1-6 branch (*) or of the a1-3 branch
(**). In 83A3, 191A3 and 527A3, nonreducing terminal b-Gal residue attaches to GlcNAc residue of the a1-6 branch (
#
) or of the
b1-4 branch (
##
).
4264 K. Ikeda et al.(Eur. J. Biochem. 269) Ó FEBS 2002

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.
REFERENCES
1. Wassarman, P.M. (1988) Zona pellucida glycoproteins. Annu.
Rev. Biochem. 57, 415–442.
2. To
¨
pfer-Petersen, E. (1999) Carbohydrate–based interactions on
the route of a spermatozoon to fertilization. Hum. Reprod. Update
5, 314–329.
3. Harris, J.D., Hibler, D.W., Fontenot, G.K., Hsu, K.T., Yurewicz,

E.C. & Sacco, A.G. (1994) Cloning and characterization of the
zona pellucida genes and cDNAs from a variety of mammalian
species: The ZPA, ZPB and ZPC gene families. DNA Seq. 4, 361–
393.
4. Litscher, E.S. & Wassarman, P.M. (1993) Carbohydrate-mediated
adhesion of eggs and sperm during mammalian fertilization.
Trends Glycosci. Glycotechn. 5, 369–388.
5. Hokke, C.H., Damm, J.B.L., Kamerling, J.P. & Vliegenthart,
J.F.G. (1993) Structure of three acidic O-linked carbohydrate
chains of porcine zona pellucida glycoproteins. FEBS Lett. 329,
29–34.
6. Hokke, C.H., Damm, J.B.L., Penninkhof, B., Aitken, R.J.,
Kamerling, J.P. & Vliegenthart, J.F.G. (1994) Structure of
O-linked carbohydrate chains of porcine zona pellucida glyco-
proteins. Eur. J. Biochem. 221, 491–512.
7. Noguchi, S. & Nakano, M. (1992) Structure of the acidic N-linked
carbohydrate chains of the 55 kDa glycoprotein family (PZP3)
from porcine zona pellucida. Eur. J. Biochem. 209, 883–894.
8. Noguchi, S., Hatanaka, Y., Tobita, T. & Nakano, M. (1992)
Structural analysis of the N-linked carbohydrate chains of the 55
kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur.
J. Biochem. 204, 1089–1100.
9. Yurewicz, E.C., Pack, B.A. & Sacco, A.G. (1991) Isolation,
composition, and biological activity of sugar chains of porcine
oocyte zona pellucida 55K glycoproteins. Mol. Reprod. Dev. 30,
126–134.
10. Kudo, K., Yonezawa, N., Katsumata, T., Aoki, H. & Nakano, M.
(1998) Localization of carbohydrate chains of pig sperm ligand in
the glycoprotein ZPB of egg zona pellucida. Eur. J. Biochem. 252,
492–499.

11. Yonezawa, N., Mitsui, S., Kudo, K. & Nakano, M. (1997)
Involvement of N-linked carbohydrate chains of pig zona pellucida
in sperm-egg binding. Eur. J. Biochem. 248,86–92.
12. Yurewicz, E.C., Sacco, A.G., Gupta, S.K., Xu, N. & Gage, D.A.
(1998) Hetero-oligomerization-dependent binding of pig oocyte
zona pellucida glycoproteins ZPB and ZPC to boar sperm mem-
brane vesicles. J. Biol. Chem. 273, 7488–7494.
13. Yonezawa, N., Fukui, N., Kudo, K. & Nakano, M. (1999)
Localization of neutral N-linked carbohydrate chains in pig zona
pellucida glycoprotein ZPC. Eur. J. Biochem. 260, 57–63.
14. Katsumata, T., Noguchi, S., Yonezawa, N., Tanokura, M. &
Nakano, M. (1996) Structural characterization of the N-linked
carbohydrate chains of the zona pellucida glycoproteins
from bovine ovarian and fertilized eggs. Eur. J. Biochem. 240, 448–
453.
15. Amari,S.,Yonezawa,N.,Mitsui,S.,Katsumata,T.,Hamano,S.,
Kuwayama, M., Hashimoto, Y., Suzuki, A., Takeda, Y. &
Nakano, M. (2001) Essential role of the nonreducing terminal
a-mannosyl residues of the N-linked carbohydrate chain of bovine
zona pellucida glycoproteins in sperm-egg binding. Mol. Reprod.
Dev. 59, 221–226.
16. Noguchi, S., Yonezawa, N., Katsumata, T., Hashizume, K.,
Kuwayama, M., Hamano, S., Watanabe, S. & Nakano, M. (1994)
Characterization of the zona pellucida glycoproteins from
bovine ovarian and fertilized eggs. Biochim. Biophys. Acta 1201,
7–14.
17. Iwamoto,K.,Ikeda,K.,Yonezawa,N.,Noguchi,S.,Kudo,K.,
Hamano, S., Kuwayama, M. & Nakano, M. (1999) Disulfide
formation in the bovine zona pellucida glycoproteins during fer-
tilization: an evidence for the involvement of cystine cross-linkages

in zona hardening. J. Reprod. Fertil. 117, 395–402.
18. Yonezawa, N., Fukui, N., Kuno, M., Shinoda, M., Goko, S.,
Mitsui, S. & Nakano, M. (2001) Molecular cloning of bovine
zona pellucida glycoproteins ZPA and ZPB and analysis for
sperm-binding component of the zona. Eur. J. Biochem. 268,
3587–3594.
19. Laemmli, U.K. (1971) Cleavage of structural protein during the
assembly of the head of bacteriophage T4. Nature (London) 227,
680–685.
20. Kosakai, M. & Yoshizawa, Z. (1982) High performance liquid
chromatography of pyridylamino derivatives of sulfated oligo-
saccharides in the deamination products of porcine and whale
heparins. J. Biochem. (Tokyo) 92, 295–303.
21. Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y. &
Takahashi, N. (1988) Analysis of N-linked oligosaccharides
using a two-dimensional mapping technique. Anal. Biochem. 171,
73–90.
22. Kornfeld, K., Reitman, M.L. & Kornfeld, R. (1981) The carbo-
hydrate-binding specificity of pea and lentil lectins. Fucose is an
important determinant. J. Biol. Chem. 256, 6633–6640.
23. Nakano, M., Yonezawa, N., Hatanaka, Y. & Noguchi, S. (1996)
Structure and function of the N-linked carbohydrate chains of pig
zona pellucida glycoproteins. J. Reprod. Fertil. Supplement 30,
25–34.
24. Nakano, M., Hatanaka, Y., Kobayashi, N., Noguchi, S., Ishikawa,
S. & Tobita, T. (1990) Further fractionation of the glycoprotein
families of porcine zona pellucida by anion-exchange HPLC and
some characterization of the separated fractions. J. Biochem.
(Tokyo) 107, 144–150.
25. Bork, P. & Sander, C. (1992) A large domain common to sperm

receptors (ZP2 and ZP3) and TGF-b type III receptor. FEBS Lett.
300, 237–240.
26. Nakano, M. & Yonezawa, N. (2001) Localization of sperm ligand
carbohydrate chains in pig zona pellucida glycoproteins. Cells
Tissues Organs 168, 65–75.
27. Prasad, S.V., Skinner, S.M., Carino, C., Wang, N., Cartwright, J.
& Dunber, B.S. (1999) Structure and function of the proteins
of the mammalian zona pellucida. Cells Tissues Organs 166,
148–164.
Ó FEBS 2002 Localization of N-linked chains in ZPA (Eur. J. Biochem. 269) 4265
28. Pepin, M C., Beauchemin, M., Plamondon, J. & O’Connor-
McCourt, M.D. (1994) Mapping of the ligand binding domain of
the transforming growth factor b receptor type III by deletion
mutagenesis. Proc. Natl Acad. Sci. USA 91, 6997–7001.
29. Derynck, R. & Feng, X H. (1997) TGF-b receptor signaling.
Biochim. Biophys. Acta 1333, F105–F150.
30. van Rooijen, J.J.M., Voskamp, A.F., Kamerling, J.P. & Vlie-
genthart, J.F.G. (1999) Glycosylation sites and site-specific gly-
cosylation in human Tamm-Horsfall glycoprotein. Glycobiology 9,
21–30.
31. Fukuoka, S. & Freedman, S.D., Yu, H., Sukhatme, V.P. &
Scheele, G.A. (1992) GP-2/THP gene family encodes self-binding
glycosylphosphatidylinositol-anchored proteins in apical secretory
compartments of pancreas and kidney. Proc. Natl Acad. Sci. USA
89, 1189–1193.
32. Chen,D.,Xu,X.,Zhu,L.,Angervo,M.,Li,Q.,Bagchi,M.K.&
Bagchi, I.C. (1999) Cloning and uterus/oviduct-specific expression
of a novel estrogen-regulated gene (ERG1). J. Biol. Chem. 274,
32215–32224.
33. Kasik, J.W. (1998) A cDNA cloned from pregnant mouse uterus

exhibits temporo-spatial expression and predicts a novel protein.
Biochem. J. 338, 947–950.
34. Legan, P.K., Rau, A., Keen, J.N. & Richardson, G.P. (1997) The
mouse tectorins. Modular matrix proteins of the inner ear
homologous to components of the sperm-egg adhesion system.
J. Biol. Chem. 272, 8791–8801.
35. Verhoeven, K., Laer, L.V., Kirschhofer, K., Legan, P.K., Hughes,
D.C., Schatterman, I., Verstreken, M., Hauwe, P.V., Couche, P.,
Chen,A.,Smith,R.J.H.,Somers,T.,Officiers,F.E.,Heyning,
P.V., Richardson, G.P., Wachtler, F., Kimberling, W.J., Willems,
P.J., Govaerts, P.J. & Camp, G.V. (1998) Mutations in the human
a-tectorin gene cause autosomal dominant non-syndromic hearing
impairment. Nat. Genet. 19,60–62.
4266 K. Ikeda et al.(Eur. J. Biochem. 269) Ó FEBS 2002