BIOLOGY OF REPRODUCTION 51, 262-272 (1994)
O-Linked Trisaccharide and N-Linked Poly-N-Acetyllactosaminyl Glycans Are Present on
Mouse ZP2 and ZP31
2
S.K. NAGDAS, Y. ARAKI, C.A. CHAYKO, M.-C. ORGEBIN-CRIST, and D.RP. TLSIANI
Centerfor Reproductive Biology Research and Department of Obstetrics & Gynecology, Vanderbilt University
School of Medicine, Nashville, Tennessee 37232-2633
ABSTRACT
Mammalian oocytes are surrounded by an extracellular glycocalyx, the zona pellucida (ZP). In the mouse, the ZP is composed
of three glycoproteins, designated mZPI, mZP2, and mZP3. Extensive studies in this species have resulted in the identification
of primary (mZP3) and secondary (mZP2) receptors for spermatozoa. In this paper we present evidence for the occurrence of
poly-N-acetyllactosaminyl glycans and an O-linked trisaccharide on mZP2 and mZP3. When exhaustively digested with endo-[galactosidase, an enzyme known to cleave repeating units of acetyllactosamine (3Gal 1,4GlcNAc 1), mZP2 and mZP3 showed
an apparent reduction in size by 23 kDa and 16 kDa, respectively. Experimental evidence included in this report indicates that
polylactosaminyl glycans are present on N-linked sugar chains. In addition, O-linked sugar chains of mZP3 have been characterized. First, treatment of de-N-glycosylated mZP3 with O-glycanase in the presence of exo-glycosidases (sialidase, a-L-fucosidase,
and N-acetylglucosaminidase) caused an apparent reduction in its size by 2-3 kDa as determined by SDS-PAGE. Second, treatment
3
of the de-N-glycosylated mZP3 with mild alkali in the presence of 1 M NaB H4 released radiolabeled oligosaccharide (OS) that
eluted from a high-resolution Bio-Gel P-4 column at the position of a trisaccharide. The radiolabeled OS had the following
structure: GlcNAc -- Gal1p,3GaINAcol. The structure was established by sizing on the Bio-Gel P-4 column, followed by examination of the susceptibility of the OS to exo-glycosidases and by its adsorbability to immobilized lectin (PNA). Potential roles
of N-linked and O-linked sugar chains in sperm-egg interaction are herein discussed.
OS chains [11]) caused reduction in its molecular mass by
30 kDa. However, the endo F treatment had no effect on
the sperm-binding activity of mZP3, which, according to
Florman and Wassarman [11], is associated with an O-linked
OS chain of an apparent molecular mass of 3.9 kDa and,
more precisely, with the -linked galactosyl residue(s) at
the nonreducing terminus of the O-linked OS unit [12]. In
a recent report, Shur and co-workers [13] presented evidence suggesting that N-acetylglucosaminyl (rather than eagalactosyl) residues on mZP3 are recognized by a galactosyltransferase (GalTase) present on mouse spermatozoa. The
sperm enzyme-zona substrate complex remains stable until
the next event in fertilization is triggered. Interestingly, it
has been reported in an abstract that treatment of zonaintact eggs with almond glycopeptidase (an endo-enzyme
that hydrolyses 3-aspartyl-glucosamine of all classes of Nlinked OS units) greatly reduces sperm-egg binding in vitro
[14]. This preliminary study implies that N-linked OS have
a role in sperm-egg interaction. We have recently reported
the presence of N-linked high-mannose/hybrid OS chain(s)
on mouse ZP2 and ZP3 [9] and presented evidence suggesting that these OS unit(s) may be a part of the recognition and binding site(s) for a sperm surface mannosidase
[15, 16].
The purpose of the present study was to further characterize the N-linked and O-linked OS chains of mZP2 and
mZP3. Our results indicate that 1) both mZP2 and mZP3
contain N-linked polylactosaminyl glycans and 2) both mZP2
and mZP3 possess O-linked trisaccharide with the structure
GlcNAc { --> Gal 31,3GalNAcol. A preliminary report on
this work has been previously presented [17].
INTRODUCTION
The mammalian egg plasma membrane is surrounded
by a thick extracellular glycocalyx, the zona pellucida (ZP).
In the mouse, the ZP is composed of three glycoconjugates,
designated mZP1, mZP2, and mZP3 [1], that are synthesized
and secreted during oocyte growth [2-6]. In recent years,
considerable progress has been made in the identification
of sperm and zona surface components believed to be responsible for gamete interaction. In particular, work on the
mouse ZP has resulted in the identification of primary (mZP3)
and secondary (mZP2) receptors for spermatozoa [7, 8]. Both
mZP2 and mZP3 are highly glycosylated and have been
shown to contain N-linked oligosaccharides (OS; 40 kDa
and 30 kDa for mZP2 and mZP3, respectively) and O-linked
(9 kDa for each of the two components) OS units. The two
ZP glycoconjugates, like other glycoproteins, show extensive microheterogeneity [3, 6, 9, 10]. Evidence accumulated
thus far strongly suggests that the sperm-binding activity of
mZP3 is associated with its OS [10].
Despite these numerous advances, considerable controversy remains regarding the precise identity of the glycan
residue(s) responsible for the ligand activity or activities.
For instance, digestion of mZP3 with endoglycosidase F (endo
F; an enzyme said to be effective in cleaving all N-linked
Accepted March 23, 1994.
Received November 12, 1993.
'This work was supported by the National Institute of Health and Human Development Grants HD25869, HD05797, and HD03820, a grant from the Andrew W.
Mellon Foundation, and research support from University Research Council.
2
Correspondence: FAX: (615) 343-7797.
262
OLIGOSACCHARIDES OF MOUSE ZONA PELLUCIDA
MATERIALS AND METHODS
Materials
Female mice (B6C3F1, 15-20 g BW) purchased from
Harlan Indust. (Indianapolis, IN) were kept under a constant 12L:12D cycle and allowed free access to food and
water. Endoglycosidase H (endo H), endo .F, N-glycanase
(recombinant peptideN-glycosidase F), and O-glycanase were
from Genzyme (Boston, MA); N-glycanase was also obtained from Oxford Glycosystems, Inc. (Rosedale, NY). Bacteriodesfragilisendo-p-galactosidase was from BoehringerMannheim Biochemicals (Indianapolis, IN). Neuraminidase
(type X), bovine epididymal a-L-fucosidase, Jack bean ]3-Nacetylglucosaminidase, and chloramine T were from Sigma
Chemical Co. (St. Louis, MO). Na' 25 I (carrier-free; 12.3 mCi/
,Ig iodine) was purchased from Amersham Corp. (Arlington Heights, IL) and was used on the day of arrival. NaB 3H4
(400 mCi/mmol) was from ICN Biochemicals, Inc. (Irvine,
CA). D-[6-3H(N)]-glucosamine-HCl (30.9 Ci/mmol) was from
Dupont Company (Boston, MA), and D-[1- 3H(N)]-galactosamine-HCI (18 Ci/mmol) was from American Radiolabeled Chemicals, Inc. (St. Louis, MO). The radiolabeled
amino sugars were converted to their alcohols by the published procedure [18] using unlabeled NaBH 4 from Aldrich
Chemical Co. (Milwaukee, WI). Chromatography paper (1
Chr) was from Whatman Ltd. (Maidstone, England).
Gal1 ,3GalNAc-ct-PNP, a synthetic substrate for O-glycanase,
was from Toronto Research Chemicals Inc. (Downsview, ON,
Canada). All electrophoretic chemicals, including marker
protein standards, gel filtration medium (Bio-Gel P-4, -400
mesh), and cation-exchange resin (AG-50W X 8, 200-400
mesh), were from Bio-Rad Laboratories (Richmond, CA).
Immobilized lectin (PNA; 4-5 mg purified from Arachis hypogaea and covalently linked per milliliter of settled agarose beads) was from E-Y Laboratories, Inc. (San Mateo, CA).
All other chemicals used were obtained commercially and
were of the highest purity available.
263
as previously described [9]. After extensive washings, the
radioiodinated material was repeatedly extracted in 1% SDS
and the solubilized zona components were resolved by SDSPAGE as earlier described [9]. The iodinated zona components were visualized by autoradiography, and each of the
three zona components (ZP1, ZP2, and ZP3) was obtained
by electroelution, exhaustive dialysis against distilled water,
and lyophilization. The whitish residue was suspended in
a small volume of distilled water (0.1-0.2 ml), divided into
small aliquots, and kept frozen (-70°C) before being used.
The zona components stored under these conditions showed
no evidence of any change in their molecular mass for at
least 4 wk.
Isolation of Unlabeled mZP Glycoconjugates
Unlabeled mZP2 and mZP3 were purified from mZP isolated from ovarian homogenates by Percoll density gradient
centrifugation [8]. Briefly, the mZP (intact and fragments)
was pelleted by centrifugation for 2 min at 13 600 rpm in
a microfuge. The pellet was suspended in 100 Rl of potassium phosphate (10 mM, pH 2.7), vortexed for 1 min, and
centrifuged as above. The supernatant was removed by aspiration and the pellet was extracted two more times as
above. The pooled supernatant (solubilized mZP) was neutralized and resolved by SDS-PAGE as described above. One
lane of the gel was stained with silver nitrate to reveal the
position of mZP1, mZP2, and mZP3. For elution of mZP2
and mZP3, their position on the unstained gel was marked
by matching with the stained gel. The marked areas were
sliced and the gel slices were subjected to electroelution
as described [9]. The electroeluted material (mZP2 or mZP3)
was exhaustively dialyzed against distilled water at 4°C, and
the dialyzed material was dried by lyophilization. The residue was suspended in 0.2 ml H20. The purity of mZP2 and
mZP3 was assessed by SDS-PAGE. Only homogeneous
preparations were used in subsequent studies. Protein was
determined by the fluorometric method [20] using BSA as
standard.
SDS-PAGE
The mouse ZP or individual zona components (before
and after treatment) were resolved by SDS-PAGE according
to Laemmli [19], using either 7.5% (ZP2) or 10% (ZP3) gel.
Other details are essentially as described [9]. The molecular
mass of mZP2 and mZP3 (before and after various treatments) was calculated on the basis of prestained high-molecular-mass (mZP2) and low-molecular-mass (mZP3) marker
proteins electrophoresed on the same gel. Owing to the
uncertain migration of glycoprotein on SDS-gel, all molecular masses reported in the present paper should be considered apparent.
Radioiodinationand Isolation of mZP Glycoconjugates
Zona-intact eggs prepared from superovulated mice were
radioiodinated by the chloramine T procedure essentially
Enzymatic Digestion of mZP2 and mZP3
Endo H, endo F, and N-glycanase digestions were carried out according to the manufacturer's instructions in the
presence of 10 ,ul toluene as described previously [21]. EndoP-galactosidase digestions were carried out by incubating
each zona component at 37°C with 5 mU of the enzyme in
a total volume of 30 ,ul containing 50 mM sodium citrate
buffer, pH 5.5, and 1.3% Nonidet P-40. Additional enzyme
(5 mU) was added after 24 h, and the mixture was incubated for an additional 24 h at 37 0C. For O-glycanase digestions, the labeled or unlabeled mZP2 and mZP3 was first
treated with N-glycanase (see above) to remove all N-linked
OS chains. The de-N-glycosylated zona components were
treated with 6 mU of O-glycanase in the presence or absence of neuraminidase (0.15 U), o-L-fucosidase (0.15 U),
264
NAGDAS ET AL.
[23] as substrate, respectively, were found to be enzymatically active.
Alkali Treatments of mZP2 and mZP3
The purified radioiodinated zona components (before
and after removal of the N-linked OS units) were subjected
to mild alkali hydrolysis (13-elimination). Since the concentration of the alkali and the treatment conditions appear to
determine the extent of cleavage of OS moieties [24, 25], in
preliminary studies we treated the intact mZP2 and mZP3
with 5, 10, 50, or 100 mM NaOH for 16 h at 37°C. Analysis
of the alkali-treated samples by SDS-PAGE showed that
whereas the NaOH at 100 mM concentration caused extensive degradation of the zona components, lower concentrations of the NaOH (5 to 50 mM) showed similar reductions in the size of mZP2 (10 kDa) and mZP3 (5 kDa). In
subsequent studies, the treatment was carried out by incubating the sample in 5 mM NaOH at 37°C for 16 h in a
total volume of 30 Il. The reaction was stopped by the addition of acetic acid to a final concentration of 5 mM. The
sample was mixed with an equal volume of twofold-concentrated sample buffer and resolved on SDS-PAGE.
FIG. 1. Assessment of OS moieties of mZP2 and mZP3. The radioiodinated ZP components purified by electroelution were treated with (lanes
2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) endo-enzymes as described
in Materials and Methods. After enzymatic digestions (endo H, lanes 1 and
2; endo F, lanes 3 and 4; endo-3-galactosidase, lanes 5 and 6; and N-glycanase, lanes 7 and 8), the samples were mixed with SDS buffer and electrophoresed under nonreducing conditions using either 7.5% gel (mZP2) or
10% gel (mZP3). The resolved components were visualized by autoradiography. The molecular masses of the prestained standard proteins electrophoresed on the same gel are shown on the left.
or N-acetylglucosaminidase (0.5 U). The digestions were
carried out at 37°C for 48 h in a total volume of 30 1 containing 10 mM sodium cacodylate buffer, pH 6.3.
GalP31,3GalNAc-ot-PNP (a synthetic substrate efficiently
cleaved by the O-glycanase used in the current study) when
incubated with endo-enzymes (N-glycanase and endo-1-galactosidase) showed no release ofp-nitrophenol even after
48 h at 37°C. Similarly, radioiodinated BSA when incubated
with the two endo-enzymes for 48 h at 37°C (see above)
showed no change in its molecular mass (data not shown).
In addition, the mZP2 and mZP3 digested with N-glycanase
in the presence of 10 mM 1,10-phenanthroline (a protease
inhibitor recommended by Genzyme) showed identical size
reductions as reported here. These results allow us to conclude that the N-glycanase and endo-p3-galactosidase preparations used in the current study are virtually free of
O-glycanase and protease activities. The commercial preparations of neuraminidase and ao-L-fucosidase when assayed
using neuramin lactose [22] andp-nitrophenyl ot-L-fucoside
Alkali Treatment of Unlabeled mZP2 and mZP3
N-Linked OS units were first removed by the exhaustive
digestion of approximately 25 g protein of each of the
mZP2 and mZP3 with 5 U of recombinant (non-glycosylated) N-glycanase. After enzymatic de-N-glycosylation, the
endo-enzyme was inactivated by heat treatment (60°C/30
min); the protein/glycoprotein backbone was precipitated
by the addition of 3 vol of ethanol and the mixture was
centrifuged as described [26]. The supernatant was removed by aspiration and the residue was washed one time
by suspension in 80% ethanol and centrifugation. The pooled
supernatant (N-linked OS units) was dried under N2 and
stored frozen at -20°C for future studies. The de-N-glycosylated mZP2 and mZP3 present in the residue were dried
and subjected to alkali hydrolysis (-elimination) by suspension of the sample in 30 Il of 5 mM NaOH containing
1 M NaB 3H4 (60 RICi/lI). After incubation at 37°C for 18 h,
the reaction was stopped by neutralization with acetic acid.
Excess NaB3H4 was removed by repeated suspension in water
and drying under N2. The final residue was suspended in
0.2 ml of water, and the sugar chains (presumably O-linked)
were separated from the protein/peptide on a column of
AG-50W (H + form) equilibrated with water [27]. The column flow-through (effluent) fractions containing radioactivity were pooled and dried in a Speed-Vac (Savant Instruments, Farmingdale, NY). The residue was suspended in 10
mM acetic acid and fractionated on a column of Bio-Gel
P-4 equilibrated with the above acetic acid [28].
Paper Chromatography
The desired fractions from the Bio-Gel P-4 column were
pooled and dried in a Speed-Vac. The residue was hydro-
265
OLIGOSACCHARIDES OF MOUSE ZONA PELLUCIDA
TABLE 1. Apparent molecular mass (in kDa) of the mZP2 and mZP3 before and after various treatments. a
mZP2
mZP3
Reduction
in kDa
Reduction
in kDa
Treatment
kDa
None
120
-
83
Endo H
Endo F
Endo--galactosidase
N-glycanaseb
120
90
97
71
0
30
23
49
83
70
67
41
0
13
16
42
41
41
41
41
41
41
38
diffuse band
0
0
0
0
0
3
de-N-glycosylated
Sialidase (S)
a-L-Fucosidase (F)
N-acetylglucosaminidase (N)
O-Glycanase
O-Glycanase + S + F
O-Glycanase + S + F + N
5 mM NaOH
71
71
71
71
71
71
71
diffuse bandc
0
0
0
0
0
0
-
kDa
c
aThe radiolabeled zona components were subjected to various treatments as described under Materials and Methods.
Following these treatments, the samples were mixed with SDS buffer and resolved on SDS-PAGE (7.5% and 10%
gel for ZP2 and ZP3, respectively) along with the prestained marker proteins. All molecular sizes reported in this
table and in various figures should be considered as apparent molecular mass.
bN-Glycanase from two sources (see Materials and Methods) gave identical results.
'Also see Figure 4. No attempt was made to calculate the molecular mass.
lysed for 6 h at 100°C in 4 M HCl [29], and the released
amino sugar alcohol was separated by cation-exchange
chromatography on a column of AG-SOW (0.5 x 6 cm)
equilibrated with water [27]. The labeled amino alditol eluted
from the column with 2 N HCl was dried under N2 and
used for paper chromatography as described elsewhere
[27,30]. Briefly, the unknown sample and the known amino
alditols ([ 3 H]glucosaminitol/[ 3H]galactosaminitol) were applied to Whatman paper (10 x 46 cm). The paper was developed in descending fashion for 16 h using the solvent
1-butanol:pyridine:water (6:4:3, v/v/v [27]) or ethyl acetate:pyridine:water (2:1:2, v/v/v [30]). The location of radioactivity was determined by cutting the paper into 1.5 x
2-cm strips and counting each strip separately in 7 ml of
Budget-Solve (Research Products International, Mount Prospect, IL). In both systems, [3H]galactosaminitol migrated more
slowly than [ 3H]glucosaminitol.
RESULTS
Evidence for Homogeneity of mZP1, mZP2, and mZP3
The purity of the electroeluted components was examined using SDS-PAGE as described in Materials and Meth-
ods. A typical autoradiogram presented in our previous report (Fig. 1 of ref. [9]) shows three distinct components of
FIG. 2. Electrophoretic analysis of mZP2 and mZP3 after treatments
with endo-p-galactosidase and/or N-glycanase. The electroeluted ZP components were treated with (endo-,-galactosidase, lane 2; N-glycanase, lane
4) or without (lane 1) endo-enzymes. After these treatments, aliquots were
withdrawn, mixed with SDS buffer, and saved for electrophoresis. The remainder of the sample was dialyzed against distilled water, lyophilized, and
then dissolved in a small volume of the distilled water. The endo-p-galactosidase-digested mZP2 and mZP3 were next treated with N-glycanase (lane
3), whereas the N-glycanase-digested sample was treated with endo-o-galactosidase (lane 5). After these digestions, the samples were resolved on
SDS-PAGE under nonreducing conditions. Other details are described in
the legend to Figure 1 and in Materials and Methods. The apparent molecular mass (kDa) of the mZP2 and mZP3 (before and after various treatments) is shown on the left.
apparent molecular weight (Mr) 200 000 (mZP1), 120 000
(mZP2), and 83 000 (mZP3). The molecular weights of the
three zona components are in agreement with findings in
several published reports [7, 13, 31, 32].
Evidence for the Presence of N-Linked Polylactosaminyl
Glycans on mZP2 and mZP3
Preliminary evidence indicated that all three zona components contain a variety of N-linked OS chains. In the
present study, experiments were carried out to characterize
OS moieties of mZP2 and mZP3, the two zona components
266
NAGDAS ET AL.
FIG. 3. Assessment of O-linked OS moieties of the mouse ZP2 and ZP3. The radioiodinated ZP components were
exhaustively digested with N-glycanase. The de-N-glycosylated mZP2 and mZP3 were dialyzed and lyophilized, and
the residue was dissolved in 60 p1of distilled water. The radiolabeled sample was divided into six equal parts and
treated without (lane 1) or with exo- and/or endo-enzyme lane 2, sialidase; lane 3, -L-fucosidase; lane 4, O-glycanase; lane 5, -N-acetylglucosaminidase; and lane 6, mixture of O-glycanase, sialidase, c(-L-fucosidase, and N-acetylglucosaminidase). Similar enzymatic digestion with a mixture of O-glycanase, sialidase, and a-L-fucosidase had no
effect on de-N-glycosylated mZP2 and mZP3 (data not included). After these treatments, the samples were mixed with
the sample buffer, resolved on SDS-PAGE, and visualized as described under Materials and Methods. Other details
are the same as in legend to Figure 1.
believed to possess ligand activity [7,8, 11]. The radiolabeled mZP2 or mZP3 was digested with or without endoglycosidases as described in Materials and Methods, mixed
with SDS-buffer, and resolved on SDS-PAGE. Results from
a typical experiment, presented in Figure 1, show the following. 1) The mobilities of both mZP2 and mZP3 were not
changed as a result of treatment with endo H (Fig. 1, lanes
1 and 2), an enzyme known to cleave high-mannose/hybrid
OS units [33]. This result is in agreement with previous observations on mouse ZP2 and ZP3 [2, 4]. However, we have
recently reported the presence of high-mannose/hybrid OS
chains on mouse ZP2 and ZP3, and have presented evidence suggesting that these OS chains may be sulfated and
are therefore insensitive to endo H treatment [9]. 2) Treatment of mZP2 and mZP3 (Fig. 1, lanes 3 and 4) with endo
F (cleaves N-linked high-mannose/hybrid and biantennary
complex-type OS chains) caused reduction in their size by
30 kDa and 13 kDa, respectively. 3) Treatment with endoP-galactosidase (cleaves repeat units of acetyllactosamine)
reduced the size of mZP2 and mZP3 (Fig. 1, lanes 5 and 6)
by 23 kDa and 16 kDa, respectively, indicating the presence
of polylactosaminyl glycans on both zona components. 4)
Enzymatic digestion with N-glycanase (cleaves all types of
N-linked OS chains) caused reduction in the size of mZP2
and mZP3 by 49 kDa and 42 kDa, respectively (Fig. 1, lanes
7 and 8). These data, summarized in Table 1, indicate that
mouse ZP2 and ZP3 contain 49 kDa and 42 kDa of N-linked
OS moieties, respectively.
Since the pig zona has been shown to contain polylactosaminyl glycans [34], we wondered whether the repeat
units of lactosaminyl residues observed on mZP2 and mZP3
are present on N-linked and/or O-linked OS chains. This
was examined in two different ways. In the first set of experiments, the radiolabeled mZP2 and mZP3 were exhaustively treated with endo-[-galactosidase to remove polylactosaminyl residues (Fig. 2, lane 2). The resulting mZP2 and
mZP3 were then subjected to N-glycanase treatment, and
the de-N-glycosylated zona components were resolved on
267
OLIGOSACCHARIDES OF MOUSE ZONA PELLUCIDA
20
10
8
6
4
z
FIG. 4. Mild alkali treatments of the de-N-glycosylated mZP2 and mZP3.
The samples were treated without (lane 1) or with (lane 2) N-glycanase (see
Materials and Methods). Aliquots (-10 000 cpm) from the N-glycanase-treated
sample were dialyzed against distilled water and lyophilized. The residue
was suspended in 5 mM NaOH and subjected to a-elimination as described
in Materials and Methods. After alkali treatment, the sample was neutralized, mixed with the sample buffer, and resolved on SDS-PAGE under nonreducing conditions (lane 3). Other details are the same as in legend to
Figure 1.
SDS-PAGE and visualized. The data presented in Figure 2
show that the sequential treatments (endo-13-galactosidase
followed by N-glycanase) reduced the size of mZP2 and mZP3
by 49 kDa and 42 kDa, respectively-values identical to those
observed when the two zona components were treated with
N-glycanase alone (Table 1). In the second set of experiments, we reversed the endo-enzyme treatments by first digesting with N-glycanase (to remove all N-linked OS chains)
and then digesting with endo-I3-galactosidase. Data from this
experiment, presented in Figure 2, lane 5, show that the
endo-p-galactosidase treatment did not cleave any additional OS chains from the two zona components. Taken together, these data suggest that the polylactosaminyl glycans
are present only on N-linked OS chains.
2
0
F=
C.)
4
LL
0
20
10
8
C
C.)
2
6
IL
4
L)
2
0
8
6
4
2
0
FRACTION NO.
3
Evidence for the Presence of O-Linked Sugar Chains on
mZP2 or mZP3
To identify O-linked OS chains on mouse ZP2 and ZP3,
the radioiodinated mZP2 and mZP3 were first treated with
N-glycanase (to remove all N-linked OS moieties) and the
resulting ZP components were treated with or without Oglycanase (an endo-enzyme known to cleave O-linked OS
chains). In addition, since the presence of terminal sugars
(such as sialyl, fucosyl, and/or glucosaminyl residues) on
O-linked OS chains is known to reduce the sensitivity of
O-glycanase [35], the enzymatic digestions were also carried out in the presence or absence of these exoglycosidases. Data from these studies, presented in Figure 3 and
Table 1, showed an apparent reduction of 2-3 kDa in the
size of mZP3 (but not mZP2) only when the incubations
were carried out in the presence of O-glycanase, sialidase,
fucosidase, and [3-N-acetyglucosaminidase. These data suggest that mZP3 contains O-linked OS.
FIG. 5. Fractionation of [ H]-labeled products formed after mild alkali
hydrolysis of de-N-glycosylated mZP2, mZP3, and fetuin. The reaction mixture contained de-N-glycosylated sample (-25 RIgprotein) in 5 mM NaOH
and 1 M NaB 3H4. After incubation for 18 h at 37°C, the reactions were stopped
by neutralizing with glacial acetic acid. Excess of NaB 3H4 was removed by
repeated drying under N2. The [3H]-labeled OS was obtained as described
in Materials and Methods, dissolved in 0.1 ml of 10 mM acetic acid, and
applied to a Bio-Gel P-4 column (1 x 212 cm, -400 mesh) equilibrated with
10 mM acetic acid. Fractions (1.2 ml) were collected and the radioactivity
was measured in aliquots. The column void volume (Ve) was determined
through use of BSA. The standards are M8 N, MansGlcNAc; MN,
Man 5GlcNAc; M3N, Man 3GlcNAc; MN 2, ManGIcNAcGIcNAc; MN, ManGIcNAc; M, Mannose.
Attempts were then made to treat the radioiodinated zona
components with mild alkali, a chemical reaction reported
to cleave O-linked OS units [25]. These studies were approached in two ways. In the first approach, radioiodinated
mZP2 and mZP3 were treated with N-glycanase, and the resulting zona components were subjected to mild alkali hydrolysis by incubation in the presence of 5 mM NaOH as
268
NAGDAS ET AL.
A
Z
UO
1
2
4,
2
1.2
-
Z
4,
4,4
A
2.8 -
1
0.
0.4
-
2
1.2 I
x
B
1
z
2
0
0
I
LL
0
'
I
I
1
B
0.8
0.4
0
1.2 -
1
C
0.8 -
V),
C.)
3
C.
0
15
30
45
DISTANCE FROM ORIGIN (CM)
FIG. 6. Identification of the radiolabeled amino sugar alcohol. The [3H]labeled trisaccharide was hydrolyzed in 4 N HCI for 6 h at 100°C [29]. Mobility of the resulting deacetylated amino sugar alcohol was compared
with the standards by paper chromatography either in solvent A
(1-butanol:pyridine:water, 6:4:3, v/v/v) or solvent B (ethylacetate:pyridine:water, 2:1:2, v/v/v/) as described in Materials and Methods.
The standards are 1) [3 H]galactosaminitol, 2) 13 H]glucosaminitol. The location of radioactivity was determined by cutting the developed and air-dried
paper into 1.5 x 2-cm strips. Each strip was mixed with 7 ml of BudgetSolve, and the radioactivity was measured by liquid scintillation spectroscopy.
described in Materialsand Methods. The alkali-treated mZP2
and mZP3 when resolved on SDS-PAGE show diffuse bands
with significant loss of the radioactivity (Fig. 4, lane 3), a
result suggesting that the polypeptide backbone of the mZP2
and mZP3 (without the N-linked OS units) is degraded by
the alkali treatment. Similar degradation of the polypeptide
has been reported for eCG and de-N-glycosylated rat zona
components [21, 36]. We then treated the intact radioiodinated mZP2 and mZP3 by incubation in 5 mM NaOH solution. The resulting zona components, when resolved on SDSPAGE, showed an apparent reduction of 5-10 kDa, a value
slightly higher than the reduction of 2-3 kDa observed after the removal of O-linked OS (see above). It is likely that
these differences are due to the uncertain migration of the
glycoconjugates on SDS-PAGE.
Next, we prepared de-N-glycosylated mZP2, mZP3, and
fetuin after exhaustive treatment with recombinant (nonglycosylated) N-glycanase. The de-N-glycosylated samples
(-25 g protein) were subjected to mild alkaline hydrolysis in the presence of NaB 3H4 as described in Materials
andMethods. Following 1-elimination, the radioactivity was
0.4 0 -
0 ..
1.2 -
1
I
I
I
100
110
, D
0.8 0.4 r~~~~
1.2 0.8
---
E
-
0.4 m
50
I
90
120
FRACTION NO.
3
FIG. 7. Susceptibility of [ H1-labeled trisaccharide to exoglycosidase or
endo-j-galactosidase. The trisaccharide obtained from mZP3 (Fig. 5, -6000
cpm) was mixed with appropriate buffer and enzyme as described in Materials and Methods. After incubation at 37'C for 48 h, the reaction was
stopped by heating the mixture at 100'C for 2-3 min. The mixture was
fractionated on a column of Bio-Gel P-4. Other details are same as described in the legend to Figure 5. Various enzymatic treatments were as
follows: (A) untreated control; (B) p-D-galactosidase; (C) -N-acetylglucosaminidase; (D) mixture of -D-galactosidase and -N-acetylglucosaminidase; (E) endo-p-galactosidase.
fractionated on a column of AG-50W X8. The radioactivity
present in the void volume (effluent) fractions was pooled,
dried in a Speed-Vac, suspended in 0.1 ml of 10 mM acetic
acid, and fractionated by gel filtration on a column of BioGel P-4 [28]. The elution profiles for mZP2, mZP3, and fetuin, presented in Figure 5, showed some similarities. For
instance, all three samples showed some radioactivity in the
void volume (Ve) and in the column inclusion volume fractions (near the position of [3H]mannose). In addition, whereas
269
OLIGOSACCHARIDES OF MOUSE ZONA PELLUCIDA
TABLE 2. Binding of labeled oligosaccharide (OS) to immobilized PNA
column.*
Radioactivity
(%Distribution)
OS from Figure 6
A
B
C
D
E
Unadsorbed
Adsorbed
100
100
29
88
86
0
0
71
12
14
*Isolated oligosaccharide from Fig. 6 (A-E, -3000 cpm) in 10 mM Tris-HCI
buffer, pH 7.2, containing 0.15 M NaCI was applied to a PNA-Agarose column (0.5 x 6 cm) equilibrated with the above buffer. After the column
was washed with 7 ml of the above buffer (flow rate, 6 ml/h), it was eluted
with the above buffer containing 0.2 M galactose. Fractions (0.5 ml) were
collected, and the radioactivity was determined. Recovery of the radioactivity in the wash (unadsorbed) and eluted (adsorbed) was quantitative.
the fetuin showed the presence of three additional radioactive peaks, the additional radioactivity from mZP2 and mZP3
eluted as a broad peak at the position of a trisaccharide
(Fig. 5). When the peak fractions were hydrolyzed in HCl
and subjected to paper chromatography as described above,
all of the radioactivity was found at the position of standard
galactosaminitol (Fig. 6), an amino sugar present at the reducing terminus of O-linked sugar chains.
The O-linked sugar chain(s) resulting from -elimination of the de-N-glycosylated mZP3 was further characterized by examining 1) the size of the sugar chain before and
after digestion with exo-enzymes and 2) the lectin-binding
properties of the OS unit before and after cleaving terminal
sugar residue(s). Data obtained via the first approach, presented in Figure 7, show that the sugar chain is sensitive
only to N-acetyglucosaminidase treatments carried out either
in the absence or in the presence of -D-galactosidase. In
both cases, the sugar chain eluted at a position smaller than
the trisaccharide. These results are in agreement with the
studies described above and confirm the presence of Nacetylglucosaminyl residue at the nonreducing terminus. In
addition, the results suggest that the amino sugar is linked
to a galactosyl residue. This finding was further confirmed
by immobilized lectin chromatography on a column of PNA
This lectin binds to GalP1,3 GalNAc only when the terminal
galactosyl residue is not covered [37]. The results presented
in Table 2 show that a significant amount of the radioactivity bound to the lectin only when the trisaccharide was
treated with N-acetylglucosaminidase. However, the sugar
chain lost affinity for the lectin when the digestion was carried out with a mixture of N-acetylglucosaminidase and 13D-galactosidase (Table 2). Taken together, the results allow
us to conclude that the major radiolabeled O-linked OS unit
has the following structure: GlcNAc
-- Gal 131,3 GalNAcol.
The radioactivity present in the void volume fractions of
the Bio-Gel P-4 column (Fig. 5) is likely to be similar to
that reported in the void volume fractions from a Bio-Gel
P-6 column [11]. The radioactive component(s) in these
fractions was insensitive to treatment with a mixture of endo(endo-13-galactosidase) and exo-(0a-D-galactosidase, sialidase, P3-D-galactosidase, and N-acetylglucosaminidase) enzymes. Moreover, hydrolysis in 4 M HCl and resolution by
paper chromatography provided no evidence for the presence of amino sugar alcohol (data not included). Thus it is
reasonable to assume that the observed radioactivity resides in the polypeptide backbone/fragments.
DISCUSSION
Direct chemical analysis of various OS chains of mouse
zona components has been rather difficult because of the
availability of very small quantities of the purified sample.
To overcome this difficulty, Bleil and Wassarman [1, 38] were
the first to radioiodinate mouse ZP. Their efforts have led
to the characterization of various components of mouse ZP
and to recognition of their potential role in sperm-egg interactions [7, 8, 10]. These early studies suggested that mZP3
is the primary ligand that binds to capacitated spermatozoa
prior to the acrosome reaction, and that mZP2 is the secondary ligand that binds to spermatozoa after the acrosome
reaction. Structural studies indicated that endo F-sensitive
N-linked OS contribute 40 kDa and 30 kDa to the molecular mass of mZP2 and mZP3, respectively [11]. In addition,
the two zona components contain 9 kDa of alkali-sensitive
OS and are presumed to be O-linked [11].
Comparison of the molecular mass of the mZP2 and mZP3
before and after N-glycanase treatment shows an apparent
reduction in their size by 49 kDa and 42 kDa, respectively.
Although it is difficult to compare the present results with
those of earlier work [11] because of uncertain migration
of glycoproteins on SDS-PAGE, it is important to comment
on the substrate specificity of the two endo-enzymes (endo
F and N-glycanase) used in the two studies. Endo F is known
to hydrolyze the glycosidic bonds of the N-N-diacetylchitobiose core structure of many high-mannose/hybrid and
biantennary complex-type asparagine-linked OS [39, 40].
Hybrid structures containing bisecting N-acetylglucosamine
linked p1,4 to the mannose core, as well as the tri- and
tetraantennary structures found in N-linked glycoproteins,
are said to be resistant to the endo F treatment [40]. The
N-glycanase used in the present study cleaves the 13-aspartyl-glucosylamine bond between asparagine and the innermost N-acetylglucosamine of the glycan of all classes of
N-linked OS chains, including high-mannose, hybrid, bi-,
tri- and tetraantennary complex type [41]. The cleaved OSs
have the chitobiose core still intact. In addition, although
the presence of phosphate, sulfate, and sialic acid groups
on the N-linked OS chains affects the activity of endo F,
these groups have no effect on the activity of the N-glycanase [39-41]. It should be noted that the endo F used by
Florman and Wassarman [11] was a gift from John H. Elder
and was presumably prepared by the published procedure
[39]. Such enzyme preparations were later shown to con-
270
NAGDAS ET AL.
tain peptide N-glycosidase F [40,41], also known as N-glycanase. The two endo-enzyme (endo F and N-glycanase)
activities have now been resolved, and they are shown to
have different substrate specificities (see above) as well as
different properties. Whereas the endo F has been shown
to be optimally active at pH 4.0-6.0, the N-glycanase used
in the present study is optimally active at pH 8.7 [40]. The
endo F (mixture of endo F and N-glycanase) treatments in
the earlier study were done at pH 6.1 [11], a value where
the N-glycanase is only half as active as at its optimal pH
[40]. Thus our observation that mouse ZP2 and ZP3 contain
10-12 KDa more N-linked OS chains than previously reported may be explained by our use of N-glycanase treatment under optimal conditions.
Although the present results have provided evidence for
the presence of O-linked polylactosaminylated glycan, it is
conceivable that this type of O-linked glycan may still be
present on mZP2 and mZP3 but is resistant to the endo-pgalactosidase or mild alkali treatments. However, the evidence that the radioactivity present in the void volume fractions in Figure 5 (the only other radioactive peak besides
the trisaccharide) contained no [3 H]galactosaminitol suggests the likelihood that the trisaccharide is the only Olinked glycan present on mZP2 and mZP3. Our results demonstrate the presence of N-linked polylactosaminyl residues
on mZP2 and mZP3. The OS chains containing repeat units
of N-acetyllactosamine (3Gal31,4GlcNAcP1) have been
demonstrated in several glycoproteins [42-49] including
porcine ZP3 [34,50]. Since the poly-N-acetyllactosamine
chains are generally present on complex-type tri- and tetraantennary N-linked OS [45], they are expected to be resistant to endo F treatments. The polylactosaminyl glycans
in the present study are shown to contribute 23 kDa and
16 kDa to mZP2 and mZP3, respectively (Table 1). These
molecular masses are based on an apparent reduction in
the size of zona components (before and after the enzyme
treatments), a criterion often used by others [11]. The presence of such a significant level of lactosaminyl glycans suggests that the two mZP glycoproteins contain a variety of
structurally variable polylactosaminyl chains. Indeed, current evidence indicates that the polylactosamine chains in
a mouse lymphoma cell line have four different terminal
sequences including a-linked galactosyl residue [48]. The
occurrence of a-linked galactosyl residues has been reported in a number of well-defined glycoconjugates, including cell-surface components [44,49, 51].
Treatment of the de-N-glycosylated mZP3 (but not mZP2)
with the O-glycanase in the presence of exo-glycosidases
showed an apparent reduction in size by 2-3 kDa (Fig. 3).
The fact that the carbohydrate chain(s) is sensitive to Oglycanase only when N-acetylglycosaminidase is included in
the digestion mixture suggests the presence of an N-acetylglucosaminyl residue on the nonreducing terminus of the
O-glycanase-sensitive OS unit(s). This was further confirmed when we subjected the de-N-glycosylated fetuin,
mZP2, and mZP3 to alkali hydrolysis (-elimination) in the
presence of NaB 3H4 . As expected, the labeled sugar chains
from fetuin, known to contain three O-linked OS chains
[52], resolved into three peaks (Fig. 5). However, the labeled sugar chain released from the two zona components
eluted in a single peak at the position of trisaccharide (Fig.
5). Data from several sets of experiments indicate that the
trisaccharide has the structure GIcNAc ---> Gal3pl,3GalNAcol.
First, the radioactivity released after acid hydrolysis of the
sugar chain comigrated with galactosaminitol, an amino sugar
found at the reducing terminus of O-linked glycoproteins
[27]. Second, the trisaccharide was sensitive to digestions
with P-N-acetylglucosaminidase alone or in combination with
1-D-galactosidase (Fig. 7). When digested with p-N-acetylglucosaminidase alone, the radioactive peak does show some
shift toward small size (compare peaks in panel A vs. panel
C). However, this is somewhat less than the shift that would
be expected if the trisaccharide were completely de-glucosaminylated. The fact that only 71% of the radioactivity
from panel C binds to the immobilized PNA lectin, and presumably has an exposed galactosyl residue, suggests that
the remaining 29% that does not bind to the lectin still
contains terminal N-acetylglucosaminyl residues (Table 2).
It is therefore reasonable to assume that the elution of the
radioactivity as a broad peak is due to the presence of a
mixture of trisaccharides (29%) and disaccharides (71%).
Combined, the evidence indicates that the N-acetylglucosaminyl residue is present at the nonreducing terminus of
the trisaccharide.
Although both mZP2 and mZP3 showed incorporation of
radioactivity in the O-linked OS, significantly higher radioactivity was incorporated into the OS unit released from
mZP3 (Fig. 5). This observation is consistent with the experimental evidence indicating that only mZP3 showed reduction in size by 2-3 kDa when treated with O-glycanase
in the presence of exo-glycosidases. The fact that the ZP3
O-linked trisaccharide structure we deduced (see above)
would have a molecular mass of 586 suggests that mZP3
may contain 4-5 sugar chains. By the same analogy, mZP2,
which showed no detectable reduction in size after digestions with O-glycanase and exo-glycosidases, may contain
fewer (1-2) sugar chains. Their contribution to the total
molecular mass is so small that their removal cannot be
evaluated on SDS-PAGE.
Our results raise questions about the chemical nature of
the nonreducing terminal residues that have been suggested to have sperm receptor activity: a-linked galactosyl
residues on 3.9-kDa O-linked OS chains [11, 12] and N-acetylglucosaminyl residues on O-linked OS chains t131. We
have found no evidence for the presence of a-linked galactosyl residues on O-linked OS units. However, N-linked
polylactosaminyl glycans are often reported to contain alinked galactosyl residues (see above). It is possible that
this sugar residue(s) is present on N-linked polylactosaminyl glycans rather than on O-linked OS chains. We have
271
OLIGOSACCHARIDES OF MOUSE ZONA PELLUCIDA
found that the O-linked trisaccharide present on mZP2 and
mZP3 has an N-acetylglucosaminyl residue on the nonreducing terminus of the molecule. It is conceivable that the
amino sugar residue(s) could be recognized by the sperm
surface galactosyltransferase as suggested by Shur and coworkers [13, 53, 54]. The presence of multiple sugar chains
on mZP3 (but not on mZP2) could explain why this zona
component acts as sperm receptor. However, this O-linked
trisaccharide chain may not be the only ligand site, since
several studies have implicated other sugar moieties of ZP
in mouse sperm-egg interactions. These include sialyl [55],
mannosyl [16,55], and N-linked OS [14] in addition to tagalactosyl [11, 12] and N-acetylglucosaminyl [13, 55, 56] residues. Since the polylactosaminyl glycans are known to possess a variety of terminal sugar residues, it is tempting to
suggest that some of the carbohydrate moieties that have
been proposed as ligands for sperm receptors may be present on the N-linked polylactosaminyl glycans we have demonstrated in the present report. Additional studies would
be needed to resolve this issue.
It is of interest that pig ZP3 (pZP3), like mZP3, is reported to contain sperm receptor activity. The pZP3 (Mr
55 000) is also highly glycosylated, containing N-linked
[34, 56], O-linked OS, including a trisaccharide [57, 58] and
poly-N-acetyllactosaminyl glycans [34, 50]. Interestingly, the
structure of the O-linked trisaccharide that contains N-acetylglucosamine on the nonreducing terminus [57] is similar
to that of the O-linked OS reported here for the mZP3. In
addition, a recent study presented structural analysis of Nlinked OS chains of pZP3 released and labeled after hydrazinolysis [56]. The labeled OS chains were separated into
neutral (28%) and acidic (72%) OS chains by anion-exchange HPLC. Evidence was presented suggesting that the
mixture of neutral N-linked glycans causes inhibition of
sperm-egg binding [56]. Also, as reported in a current abstract, the N-linked OS chains cleaved from sea urchin egg
jelly coat with N-glycanase have ligand activity and acrosome reaction-inducing activity [59]. Since the jelly coat is
analogous in many ways to the mammalian ZP, it is reasonable to assume that an N-linked OS moiety or moieties
may be involved in sperm recognition in invertebrates as
well as in mammals.
The identification of several receptors and ligands on
mammalian spermatozoa and ZP, respectively (for review
see ref. [60]), suggests that perhaps several receptor-ligand
interactions must occur before successful binding and fertilization. It is likely that the precise order or dominance
of these interactions among various mammalian species
contributes to relative species specificity. To the best of our
knowledge, the N-linked polylactosaminyl glycans of mZP2
and mZP3 have never been chemically characterized and
the presence of an O-linked trisaccharide has never been
reported. It will be important to examine the structuralfunctional relationships of both N-linked and O-linked sugar
chains present on mZP2 and mZP3. Taken together, these
studies will further our understanding of the molecular
mechanisms of gamete interactions and fertilization.
ACKNOWLEDGMENTS
The excellent secretarial assistance of Ms. Pamela Reed and Mrs. Loreita Little is
greatly acknowledged. We are indebted to Drs. Oscar Touster and Gary Olson for a
critical reading of the manuscript.
REFERENCES
1. Bleil JD, Wassarman PM. Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse oocyte's zona pellucida.
Dev Biol 1980; 76:185-203.
2. Bleil JD, Wassarman PM. Synthesis of zona pellucida proteins by denuded and
follicle-enclosed mouse oocyte during culture in itro. Proc Natl Acad Sci USA
1980; 77:1029-1033.
3. Greve JM, Salzmann GS, Roller RJ, Wassarman PM. Biosynthesis of the major zona
pellucida glycoprotein secreted by oocytes during mammalian oogenesis. Cell
1982; 31:749-759.
4. Salzmann GS, Greve JM, Roller RJ, Wassarman PM. Biosynthesis of the sperm
receptor during oogenesis in the mouse. EMBO J 1983; 2:1451-1456.
5. Roller RJ, Wassarman PM. Role of asparagine-linked oligosaccharides in secretion
of glycoproteins of the mouse egg's extracellular coat. J Biol Chem 1983; 258:1324313249.
6. Shimizu S, Tsuji M, Dean J. In vitro biosynthesis of three sulfated glycoproteins
of murine zonae pellucidae by oocytes grown in follicle culture. J Biol Chem
1983; 258:5858-5863.
7. Bleil JD, Wassarman PM. Sperm-egg interactions in the mouse: sequence of events
and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev
Biol 1983; 95:317-324.
8. Bleil JD, Wasssarman PM. Autoradiographic visualization of the-mouse egg's sperm
receptor bound to sperm. J Cell Biol 1986; 102:1363-1371.
9. Tulsiani DRP, NagDas SK, Cornwall GA, Orgebin-Crist M-C. Evidence for the presence of high-mannose/hybrid oligosaccharide chain(s) on the mouse ZP2 and
ZP3. Biol Reprod 1992; 46:93-100.
10. Wassarman PM. Cell surface carbohydrate and mammalian fertilization. In: Fukuda M (ed.), Cell Surface Carbohydrate and Development. Boston: CRC Press;
1992: 215-238.
11. Florman HM, Wassarman PM. O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity. Cell 1985; 41:313-324.
12. Bleil JD, Wassarman PM. Galactose at the nonreducing terminus of O-linked oligosaccharides of mouse egg zona pellucida glycoprotein ZP3 is essential for the
glycoprotein's sperm receptor activity. Proc Natl Acad Sci USA 1988; 85:67786782.
13. Miller DJ, Macek MB, Shur BD. Complementarity between sperm surface ,-1,4galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding. Nature 1992;
357:589-593.
14. Yamagata T. The role of saccharides in fertilization of the mouse. Dev Growth
&Differ 1985; 27:176-177.
15. Tulsiani DRP, Skudlarek MD, Orgebin-Crist M-C. Novel ca-D-mannosidase of rat
sperm plasma membranes: characterization and potential role in sperm-egg interactions. J Cell Biol 1989; 109:1257-1267.
16. Cornwall GA, Tulsiani DRP, Orgebin-Crist M-C. Inhibition of the mouse sperm
surface a-D-mannosidase inhibits sperm-egg binding in vitr. Biol Reprod 1991;
44:913-921.
17. NagDas SK, Araki Y, Chayko CA, Orgebin-Crist M-C, Tulsiani DRP. Presence of
poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from mouse
ZP2 and ZP3. J Cell Biol 1991; 112(suppl):3 (abstract 382).
18. Takasaki S, Kobata A Microdetermination of individual neutral and amino sugars
and N-acetyl-neuraminic acid in complex saccharides. J Biochem 1974; 76:783789.
19. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970; 227:680-685.
20. Anderson PM, Desnick RJ. Purification and properties of 8-aminolevulinate dehydrase from human erythrocytes. J Biol Chem 1979; 254:6924-6930.
21. Araki Y, Orgebin-Crist M-C, Tulsiani DRP. Qualitative characterization of oligosaccharide chains present on the rat zona pellucida glycoconjugates. Biol Reprod
1992; 46:912-919.
22. Tulsiani DRP, Carubelli R Studies on the soluble and lysosomal neuraminidases
of rat liver. J Biol Chem 1970; 245: 1821-1827.
272
NAGDAS ET AL.
23. Carlsen RB, Pierce JG. Purification and properties of an ct-L-fucosidase from rat
epididymis. J Biol Chem 1972; 247:23-32.
24. Iyer RN, Carlson DM. Alkaline borohydride degradation of blood group H substance. Arch Biochem Biophys 1971; 142:101-105.
25. Spiro RG, Bhoyroo VD. Structure of the O-glycosidically linked carbohydrate units
of fetuin. J Biol Chem 1974; 119:5704-5717.
26. Tulsiani DRP, Broquist HP, James LF, Touster O. The similar effects of swainsonine and locoweed on tissue glycosidases and oligosaccharides of the pig indicates that the alkaloid is the principal toxin responsible for the induction of
locoism. Arch Biochem Biophys 1984; 232:76-85.
27. Aminoff D, Gathmann WD, McLean CM, Yodomae T. Quantitation of oligosaccharides released by the 13-elimination reaction. Anal Biochem 1980; 101:44-53.
28. Tulsiani DRP, Touster O. Substrate specificities of rat kidney lysosomal and cytosolic a-D-mannosidases and effects of swainsonine suggest a role of the cytosolic enzyme in glycoprotein catabolism. J Biol Chem 1987; 262:6506-6514.
29. Tulsiani DRP, Skudlarek MD, NagDas SD, Orgebin-Crist M-C. Purification and
characterization of rat epididymal fluid a-D-mannosidase: similarities to sperm
plasma-membrane a-D-mannosidase. Biochem J 1993; 290:427-436.
30. Cabib E, Leloir LF, Cardini CE. Uridine diphosphate acetylglucosamine. J Biol
Chem 1953; 203:1070-2155.
31. East IJ, Gulyas BL, Dean J. Monoclonal antibodies to the murine zona pellucida
protein with sperm receptor activity: effects of fertilization and early development. Dev Biol 1985; 109:268-273.
32. Leyton L, Saling P. 95 kd sperm proteins bind ZP3 and serve as tyrosine kinase
substrates in response to zona binding. Cell 1989; 57:1123-1130.
33. Tarentiono AL, Maley F. Purification and properties of an endo-3-N-acetylglucosaminidase from Streptomyces grieus. J Biol Chem 1974; 249:811-817.
34. Yurewicz EC, Sacco AG, Subramanian MG. Structural characterization of the Mr
= 55 000 antigen (ZP3) of porcine oocyte zona pellucida; purification and characterization of a-and -glycoproteins following digestion of lactosaminoglycan
with endo-3-galactosidase. J Biol Chem 1987; 262:564-571.
35. Umemoto J, Bhavananden VP, Davidson EA. Purification and properties of an
s pneumoniae. J Biol Chem
endo-a-N-acetyl-Dgalactosaminidase from Diplo
1977; 252:8609-8614.
36. Bahl OP, Wagh PV. Characterization of glycoproteins: carbohydrate structures of
glycoprotein hormones. In: Dhindsa DS, Bahl OP (eds.), Molecular and Cellular
Aspects of Reproduction. New York: Plenum Press; 1986: 1-51.
37. Lotan R, Beattie G, Hubbell 1W, Nicolson GL. Activities of lectins and their immobilized derivatives in detergent solutions. Implications on the use of lectin
affinity chromatography for the purification of membrane glycoproteins. Biochemistry 1977; 16:1787-1794.
38. Bleil JD, Wassarman PM. Mammalian sperm-egg interaction: identification of a
glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm.
Cell 1980; 20:873-882.
39. Elder JH, Alexander S. Endo-l-N-actylglucosaminidase F: endoglycosidase from
Flavobacteriummeningosepticum that cleaves both high-mannose and complex
glycoproteins. Proc Natl Acad Sci USA 1982; 79:4540-4544.
40. Plummer TH Jr, Elder JH, Alexander S, Phelan AW, Tarentino AL. Demonstration
of peptide: N-glycosidase F activity in endo-1-acetylglucosaminidase F preparatons. J Biol Chem 1984; 259:10700-10704.
41. Tarentino AL, Gomez CM, Plummer TH Jr. Deglycosylation of asparagine-linked
glycans by peptide: N-glycosidase F. Biochemistry 1985; 24:4665-4671.
42. Cummings RD, Komfeld S. The distribution of repeating (Galpl1, 4 GIcNAcf1I,3)
sequence in asparagine-linked oligosaccharides of the mouse lymphoma cell lines
PW 5147 and PHAR 2.1. J Biol Chem 1984; 259:6253-6260.
43. Fukuda M, Spooncer E, Oates JE, Dell A, KlockJC. Structure of sialylated fucosyl
lactosaminoglycan isolated from human granulocytes. J Biol Chem 1984; 259:1092510935.
44. Spiro RG, Bhoyroo VD. Occurrence of ao-D-galactosyl residues in the thyroglobulins from several species. J Biol Chem 1984; 259:9858-9866.
45. Fukuda M. Cell-surface glycoconjugates as onco-differentiation markers in hematopoietic cells. Biochem Biophys Acta 1985; 780:119-150.
46. Fukuda MN, Dell A, Oates JE, Fukuda M. Embryonal lactosamininoglycan: the
structure of branched lactosaminoglycans with novel disialosyl (sialyl a2- 9 sialyl) terminals isolated from PAI human embryonal carcinoma cells. J Biol Chem
1985; 260:6623-6631.
47. Edge ASB, Spiro RG. Thyroid Cell surface glycoproteins: nature and disposition
of carbohydrate units and evaluation of their group I activity. J Biol Chem 1985;
260:15332-15337.
48. Merkle RK, Cummings RD. Relationship of the terminal sequences to the length
of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the
mouse lymphoma cell line BW 5147. J Biol Chem 1987; 262:8179-8189.
49. Okada Y, Spiro RG. Isolation and characterization of three major glycoproteins
from thyroid plasma membranes. J Biol Chem 1980; 255:8865-8872.
50. Mori E, Takasaki S, Hedrick JL, Wardrip NJ, Mori T, Kobata A. Neutral oligosaccharide structures linked to asparagine of porcine zona pellucida glycoproteins.
Biochemistry 1991; 30:2078-2087.
51. Eckhardt AE, Goldstein IJ. Occurrence of a-D-galactosyl-containing glycoproteins
on ehrlich tumor cell membranes. Biochemistry 1983; 22:5280-5289.
52. Edge AS, Spiro RG. Presence of an O-glycosidically linked hexasaccharide in fetuin. J Biol Chem 1987; 262:16135-16141.
53. Shur BD, Hall NG. A role for mouse sperm surface galactosyltransferase in sperm
binding to the egg zona pellucida. J Cell Biol 1982; 95:574-579.
54. Lopez LC, Bayna EM, Litoff D, Shaper NL, Shaper JH, Shur BD. Receptor function
of mouse sperm surface galactosyltransferase during fertilization. J Cell Biol 1985;
101:1501-1510.
55. Lambert H. Role of sperm-surface glycoproteins in gamete recognition in two
mouse species. J Reprod Fertil 1984; 70:281-284.
56. Noguchi S, Hatanaka Y, Tobita T, Nakano M. Structural analysis of the N-linked
carbohydrate chains of the 55-kDa glycoprotein family (pZP3) from porcine zona
pellucida. Eur J Biochem 1992; 204:1088-1100.
57. Hirano T, Takasaki S, Hedrick JL, Wardrip NT, Amano J, Kobata A. O-linked neutral sugar chains of porcine zona pellucida glycoproteins. Eur J Biochem 1993;
159:763-769.
58. Hokke CH, Damm JBL, Kamberling JP, Vliegenthart JFG. Structure of three acidic
O-linked carbohydrate chains of porcine zona pellucida glycoproteins. FEBS Lett
1993; 329:29-34.
59. Keller SH, Vacquier VD. A glycoprotein with active N-linked oligosaccharide induces the acrosome reaction in sea urchin sperm. FASEB J 1993; 7:(abstract 1156).
60. O'Rand MG. Sperm-egg recognition and barriers to interspecies fertilization. Gamete Res 1989; 19:315-326.