From the Department of Obstetrics & Gynecology, Wayne
State University, Detroit, Michigan 48201, the ¶ Gamete Antigen
Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New
Delhi, India, and the
Department of Biochemistry, Michigan State
University, East Lansing, Michigan 48824
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ABSTRACT |
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The zona pellucida surrounding the pig oocyte contains two Mr 55,000 glycoproteins, pZPB and pZPC, which are orthologues of mouse zona proteins ZP1 and ZP3, respectively. We previously reported that isolated boar sperm membrane vesicles possess high affinity binding sites for partially purified pZPB, but not pZPC. Interestingly, co-incubation experiments also implicated pZPB-pZPC complexes as potential ligands. We now report that when depleted of a minor pZPC contaminant by size exclusion chromatography, pZPB lacks independent binding activity. In solid phase binding assays employing immobilized boar sperm membranes, pZPB failed to compete with biotin-(pZPB+pZPC) probe, and biotin-labeled pZPB yielded negligible binding. However, when co-incubated with pZPC prior to the binding assays, pZPB acted as a potent competitor, and biotin-labeled pZPB exhibited high affinity, saturable binding. Binding activity was attributed to pZPB-pZPC heterocomplexes, which were detected in co-incubation mixtures by size exclusion chromatography and Western blot analysis. In the pig, therefore, sperm membranes possess a zona-binding protein with high affinity sites for pZPB-pZPC heterocomplexes, but not free glycoprotein subunits. Consequently, associative interactions between zona molecules can contribute toward both the assembly of the zona matrix and generation of ligands important for sperm-zona interactions.
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INTRODUCTION |
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The zona pellucida is a morphologically discrete, extracellular matrix which envelops and protects the oocyte and preimplantation embryo. This egg investment also actively participates in the fertilization process. The zona pellucida provides docking sites for species-specific sperm attachment and induces bound sperm to undergo the acrosome reaction, the preparatory event for zona penetration and eventual sperm-egg fusion. This intimate association of male and female gametes is mediated in part by sperm-adhesive glycoproteins within the zona matrix and complimentary zona-binding proteins on the sperm surface. Interestingly, although multiple gamete adhesion molecules have been characterized at the molecular level (1-11), the relative physiological importance of many remains a topic of considerable debate (12).
Zonae pellucidae contain a repertoire of glycoproteins encoded by three gene families that are conserved across species. Adopting the terminology of Harris et al. (13), the three cDNA-predicted precursor polypeptides in the pig (10, 13, 14) are herein designated pZPA (79 kDa), pZPB (59 kDa), and pZPC (46 kDa). Due to post-translational processing, the three mature glycoproteins from isolated zonae appear on nonreducing SDS gels as two diffuse bands (15); pZPA runs at Mr 90,000, whereas pZPB and pZPC co-migrate at Mr 55,000. Electrophoretic or chromatographic protocols permit isolation of co-purified pZPB and pZPC glycoproteins (15-17) and such pZP(B+C) preparations exhibit several important biological activities, including: stimulation of the acrosome reaction in capacitated boar sperm (18), inhibition of sperm-zona attachment (19, 20), binding to a sulfated polysaccharide-binding domain on boar sperm proacrosin (21), and binding to isolated boar sperm membrane vesicles (22).
Although intractable to further purification as fully glycosylated
entities, pZPB and pZPC elute as separate peaks during reverse phase
HPLC1 when pretreated with
endo--galactosidase (17). The pZPC glycoprotein (Mr 41,000 following endo-
-galactosidase
digestion) elutes first and appears homogeneous on SDS gels. The pZPB
glycoprotein (Mr 44,000 following
endo-
-galactosidase digestion) follows and routinely contains small
amounts (5-10%) of pZPC contaminant.
Purified pZPB and pZPC differ markedly in apparent biological activity. When tested in gamete adhesion assays, pZPB inhibits sperm-zona binding, whereas pZPC is without effect (20, 23). Likewise, when tested in solid phase binding assays, biotin-labeled pZPB binds to immobilized boar sperm membranes in a concentration-dependent, saturable manner, whereas biotin-pZPC does not (22). In competition assays, pZPB blocks binding of a biotin-pZP(B+C) probe to membranes; pZPC fails to compete but, surprisingly, enhances the apparent affinity of pZPB for its cognate zona-binding protein. Collectively, the data suggest that (i) boar sperm possess a zona-binding protein with a pZPB-binding domain that recognizes both pZPB and putative pZPB-pZPC complexes, and (ii) interaction of pZPB with pZPC enhances pZPB binding affinity.
More recently, both pZPB- and pZPC-specific antibodies were unexpectedly found to block binding of biotin-pZPB to boar sperm membranes.2 This paradoxical data prompted us to question whether the pZPB glycoprotein indeed binds independently to membrane-associated zona-binding proteins. Could minor amounts of putative pZPB-pZPC complexes account for the observed binding activity of our previous pZPB preparations? Here we conclusively demonstrate that whereas pZPC and more highly purified pZPB lack independent binding activity, reconstituted pZPB-pZPC heterocomplexes are novel, high affinity ligands for zona-binding molecules present in boar sperm membrane vesicles.
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EXPERIMENTAL PROCEDURES |
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Materials--
Three different pig zona protein preparations
were used in this study. Their nomenclature conforms with that adapted
by Harris et al. (13). pZP(B+C) was purified from
heat-solubilized porcine zonae by gel filtration and hydroxyapatite
chromatography and consists of fully glycosylated pZPB and pZPC
glycoproteins (17). pZPB and pZPC were isolated from
endo--galactosidase-digested pZP(B+C) by reverse phase HPLC (17).
pZP(B+C), pZPB, and pZPC correspond to preparations designated ZP3,
ZP3
, and ZP3
in prior publications from this laboratory. Membrane
vesicles were isolated from capacitated boar sperm by nitrogen
cavitation and differential centrifugation and stored at
70 °C as
described previously (22).
Antibodies-- The rabbit polyclonal antiserum raised against the keyhole limpet hemocyanin-conjugated peptide CELQIAKDERYGS (residues 319-330 of the deduced pZPB precursor protein with an added N-terminal cysteine) was described previously (24). Affinity purification on a matrix consisting of the synthetic peptide coupled to AminoLink resin (Pierce) yielded pZPB-specific, site-directed antibodies (anti-pZPB319-330). For pZPC-specific immunodetection, we used an ascites fluid containing a previously described (25) mouse monoclonal antibody, MA-451, which recognizes an internal pZPC epitope (EEKLVF, residues 166-171 of cDNA-derived pZPC precursor protein) (26). Horseradish peroxidase-conjugated secondary antibodies were obtained from Amersham.
Size-exclusion Chromatography--
HPLC separations were
performed on a Dionex instrument equipped with a TSK
G3000SWXL column (7.8 × 300 mm, TosoHaas). The flow
of high-salt PBS (50 mM sodium phosphate, pH 6.6, 300 mM NaCl) was maintained at 0.5 ml/min at ambient
temperature. For preparative isolations, lyophilized proteins were
reconstituted in high-salt PBS and centrifuged through Spin-X filter
units (0.45-µm cellulose acetate, Costar) prior to injection.
Individual peaks were collected manually, pooled from multiple runs,
concentrated by ultrafiltration (Centricon-10, Amicon) to a final
protein concentration of >500 µg/ml, and stored at 20 °C. The
TSK G3000SWXL column was calibrated daily using blue
dextran 2000 and dinitrophenyl-glycine as void volume
(Vo) and total volume (Vt)
markers, respectively. The elution position (Ve) of
individual peaks was used to calculate Kav using
the equation, Kav = (Ve
Vo)/(Vt
Vo).
Mass Spectrometry-- MALDI-MS of zona proteins was performed on a PerSeptive Biosystems Voyager Elite instrument equipped with a nitrogen laser emitting at 337 nm with a pulse duration of 2 ns. The instrument was operated at an accelerating voltage of 30 KV in the linear continuous extraction mode. Sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) was used as the MALDI matrix and dissolved in acetonitrile/water/trifluoroacetic acid (50:49.9:0.1, v/v/v) to make a solution of 10 mg/ml. Proteins were dialyzed against 25 mM ammonium acetate and lyophilized to dryness prior to analysis. To prepare the target, 1 µl of the protein sample dissolved in water was mixed with 1 µl of matrix solution on the sample plate cell and air-dried.
Immunoblotting-- Zona proteins were separated by SDS-gel electrophoresis on nonreducing 10% polyacrylamide gels and transferred to nitrocellulose (Novex). After treatment with BlockerTMBLOTTO (Pierce), membranes were incubated with either 0.5 µg/ml anti-pZPB319-330 or MA-451 diluted 1:5000. Bound antibodies were decorated with horseradish peroxidase-conjugated second antibodies diluted 1:5000 and visualized with ECL reagents and Hyperfilm-ECL (Amersham).
Solid Phase Binding Assays-- Interactions between purified zona proteins and boar sperm membrane vesicles were investigated using a previously described solid phase binding assay (22) with minor modifications. Immulon-2 microtiter wells (Dynatech) were coated with 500 ng of sperm membranes and subsequently blocked with 1% BSA. For competition binding assays, wells were incubated in quadruplicate with 50 µl of 0.2 µg/ml biotin-pZP(B+C) in TBS/T/BSA (20 mM Tris, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.05% Tween 20, 0.1% BSA) in the presence and absence of unlabeled competitor. Competitors were preincubated overnight at 4 °C in high-salt PBS, diluted in TBS/T/BSA to twice their final concentration, and mixed with an equal volume of 0.4 µg/ml biotin-pZP(B+C) at least 30 min prior to transfer to wells. Binding of biotin-labeled probe was measured using strepavidin-alkaline phosphatase (Jackson ImmunoResearch) and p-nitrophenyl phosphate. Absorbance values at 405 nm were recorded using a Bio-Tek microplate reader. All data in competition assays were corrected for nonspecific binding as determined in wells incubated with biotin probe in the presence of a 100-fold excess of pZP(B+C). IC50 (mean ± S.E.) values are defined as the concentration of competitor required to inhibit binding of biotin-pZP(B+C) by 50%; for co-incubates, IC50 values represent the concentration of the pZPB component only.
Direct binding assays followed a similar protocol, except that biotin-labeled proteins were preincubated overnight at 4 °C atMiscellaneous--
For biotin labeling, 450 µg of protein was
reacted with 30 µg of D-biotinoyl--aminocaproic
acid-N-hydroxysuccinimide ester in 180 µl of 50 mM sodium borate buffer, pH 8.0. After a 1-h incubation at
ambient temperature, the reaction was quenched with ammonium chloride.
Biotinylated protein was recovered by HPLC on a TSK G3000SWXL column in high-salt PBS and concentrated by
ultrafiltration. Proteins were quantitated using a BCA Protein Assay
kit (Pierce) in a microtiter plate format. BSA was used to construct
standard curves. Protein concentrations were converted to molarities
using molecular masses of 36,670 and 36,308 Da for the core
polypeptides of pZPB (cDNA-deduced residues 137-446) and pZPC
(cDNA-deduced residues 23-348), respectively; N termini have been
experimentally determined (17, 27) and C termini are positioned at
consensus sites for furin cleavage (10, 13).
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RESULTS |
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Purification of pZPB Glycoprotein by Size Exclusion
Chromatography--
To produce a preparation depleted of residual
pZPC, we subjected pZPB to size exclusion chromatography on a TSK
G3000SWXL column in high-salt PBS. Under these conditions,
pZPB eluted as a series of incompletely resolved UV peaks (Fig.
1A). The first peak emerged in
the column void volume (exclusion limit >700 kDa) and the final peak
eluted between the 43- and 80-kDa molecular mass markers. Five
subfractions, designated pZPB1-pZPB5 and
containing 15, 17, 21, 21, and 26% of recovered protein, respectively,
were collected (Fig. 1A) and concentrated by
ultrafiltration. Macromolecular compositions were examined by
SDS-polyacrylamide gel electrophoresis and immunoblotting. Protein
staining revealed the pZPB glycoprotein as a predominant
Mr 44,000 band (95% of total Coomassie Blue staining) in the pZPB preparation used as starting material (Fig. 1B, lane B) and in each pZPB subfraction obtained
by size exclusion chromatography (Fig. 1B, lanes
1-5). By comparison, purified pZPC glycoprotein migrates as a
Mr 41,000 band (Fig. 1B, lane
C). Subfractions pZPB1-pZPB3 also
contained a Mr 81,000 band accounting for <5% of total Coomassie Blue staining.
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Competitive Binding Activity of pZPB Subfractions-- To assess relative ligand activity of pZPB subfractions toward boar sperm zona-binding proteins, we constructed competitive binding curves using immobilized membrane vesicles as the source of zona-binding proteins and biotin-pZP(B+C) as labeled probe. Significantly, only those subfractions possessing both pZPB and pZPC glycoproteins demonstrated competitor activity (Fig. 2A). Subfractions pZPB1-pZPB3 blocked binding of biotin-pZP(B+C) to immobilized membranes with respective IC50 values (n = 2) of 20.7 ± 7.1, 36.0 ± 4.9, and 103 ± 22 nM. In striking contrast, pZPB4 and pZPB5 did not compete (IC50 > 1 µM) with the biotin-pZP(B+C) probe.
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Purified pZPB5 Is Predominantly Monomeric-- We used MALDI-MS and size exclusion chromatography to evaluate the oligomeric status of purified pZPB5. The MALDI spectra of pZPB5 (Fig. 3) and pZPB starting material (not shown) were highly similar and indicated masses centered about 43,643 and 43,577 Da for the constituent families of pZPB glycoforms, respectively. However, in contrast to the complex elution profile of pZPB starting material in Fig. 1A, size exclusion chromatography of pZPB5 on the G3000SWXL column in high-salt PBS yielded a dominant peak at Kav = 0.435 (Fig. 4A); the molecular size relative to protein standards (Fig. 4B) was Mr 55,400. As this approximates the molecular mass determined by MALDI-MS, pZPB5 most likely exists as monomers in high-salt PBS. The presence of minor Mr 124,000 and 230,000 peaks at Kav = 0.313 and 0.221, respectively, indicated a tendency of the pZPB glycoprotein to self-associate.
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pZPB5 and pZPC Form Heteromultimeric Complexes-- Size exclusion chromatography provided direct evidence that co-incubated pZPB and pZPC glycoproteins form heterocomplexes. Proteins were incubated overnight at 4 °C in 40 µl of high-salt PBS as follows: (i), pZPB5, 16 µg; (ii), pZPB5, 16 µg + fetuin, 12 µg; (iii), pZPB5, 16 µg + pZPC, 12 µg; (iv), pZPC, 12 µg; and (v), pZPC, 12 µg + fetuin, 16 µg. Fetuin, a glycoprotein with physicochemical characteristics similar to pZPB and pZPC, served as a negative control. Twenty-µl aliquots were injected onto the TSK G3000SWXL HPLC column and 1-min fractions were collected. Elution of pZPB and pZPC glycoproteins was monitored by Western blotting using anti-pZPB319-330 and MA-451, respectively. When preincubated alone (not shown) or with fetuin (Fig. 5A), the pZPB5 glycoprotein eluted as a monomer in fraction 17 (cross-reference, Fig. 4A). In contrast, the pZPC glycoprotein eluted as multiple peaks (Mr 300,000, Kav = 0.178; Mr 160,000, Kav = 0.274; Mr 68,000, Kav = 0.404) in fractions 14-17 whether preincubated alone (not shown) or in the presence of fetuin (Fig. 5C); the Mr 160,000 peak (fraction 15) was predominant. Importantly, when pZPB5 and pZPC were co-incubated, the chromatographic behavior of each changed dramatically (Fig. 5, B and D); a major percentage of each glycoprotein now eluted in the void volume (fraction 11). The elution shifts demonstrate that when incubated together, pZPB and pZPC glycoproteins assemble into high mass heterocomplexes.
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High Affinity Binding of Biotin-labeled pZPB5-pZPC
Heterocomplexes to Boar Sperm Membrane Vesicles--
To further test
specificity of membrane-associated zona-binding proteins for pZPB-pZPC
heterocomplexes and to estimate binding affinities, we performed direct
binding experiments using biotin-labeled heterocomplexes generated by
co-incubation of either biotin-pZPB5 + pZPC or
pZPB5 + biotin-pZPC. In the first set of experiments, a
fixed concentration (100 µg/ml) of biotin-pZPB5 was
preincubated (overnight, 4 °C) in high-salt PBS with or without
variable quantities of pZPC. Co-incubates were diluted to yield 0.2 µg/ml biotin-pZPB5 (5.4 nM) and incubated
with immobilized membrane vesicles. Binding of biotin-pZPB5
depended strongly upon prior incubation with pZPC. At
pZPC:biotin-pZPB5 ratios <0.3:1, binding of
biotin-pZPB5 was directly proportional to the concentration
of co-incubated pZPC (Fig.
6A). Binding of
pZPC:biotin-pZPB5 co-incubates with ratios >0.3:1
plateaued at a level 30-fold higher than that observed with the
biotin-pZPB5 incubated alone sample. Presumably, under these experimental conditions, pZPC co-incubated with
biotin-pZPB5 in ratios 0.3:1 maximizes recruitment of
biotin-pZPB5 into heterocomplexes recognized by
membrane-associated zona-binding proteins. Co-incubates with
pZPC:biotin-pZPB5 or fetuin:biotin-pZPB5 ratios
of 1:1 were used to construct saturation binding curves. As shown in
Fig. 6B, pZPC-(biotin·pZPB5) heterocomplexes
exhibited high affinity, saturable binding; the EC50 value
(n = 3) equaled 4.3 ± 0.6 nM when
calculated on the basis of biotin-pZPB5 concentration. High affinity binding was notably absent following co-incubation of biotin-pZPB5 with fetuin.
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DISCUSSION |
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Using highly purified glycoproteins isolated from the pig oocyte zona pellucida, we demonstrate for the first time that pZPB and pZPC associate to form high mass heteromultimeric complexes. We also provide the first description of a zona binding activity in boar sperm membranes that possesses avid affinity for pZPB-pZPC heterocomplexes but not the free zona glycoprotein subunits. As a consequence, we propose that at least for the porcine system, pZPB-pZPC heteromers are important functional and structural units of this gamete-specific extracellular matrix.
Data presented earlier (22) suggested that pZPB binds directly to isolated boar sperm membranes and hinted that pZPB can associate with pZPC to form putative pZPB-pZPC complexes with enhanced binding affinity. Our new data obtained with pZPB depleted of residual pZPC contaminant by size exclusion chromatography confirm the heterotypic association of co-incubated pZPB and pZPC glycoproteins but conclusively demonstrate that free pZPB subunits fail to engage membrane-associated zona-binding proteins. Consequently we conclude that pZPB-pZPC complexes, although present in minor amounts, accounted exclusively for the ligand activity observed for earlier pZPB preparations (20, 22).
Size exclusion chromatography and Western blot analyses reveal that
co-incubated pZPB5 and pZPC assemble into high mass
heterocomplexes. Although the domains mediating this associative
interaction are not yet identified, pZPB residues 142-187 encompass a
cysteine-rich trefoil motif, or P-domain, which is expressed in mucins
and secretory peptides and is postulated to mediate protein-protein and
protein-carbohydrate interactions (28-30). In addition, pZPB (residues
190-454), pZPC (residues 45-306), and pZPA (residues 368-623) each
contain a single ZP domain (31) consisting of a module with eight
conserved cysteines and presumed characteristic three-dimensional
structure which has been suggested to mediate binding activities
between proteins that form filamentous extracellular matrices (32, 33). Native pig zona pellucida glycoproteins also possess N- and
O-linked oligosaccharides bearing sulfated polylactosamines
(34-36). As the pZPB and pZPC molecules have by necessity been exposed
to endo--galactosidase during the purification protocol, we can conclude that the polylactosamines associated with native glycoproteins are not required for assembly of pZPB-pZPC heterocomplexes or subsequent high affinity binding to membrane vesicles. Congruently, enzymatic removal of polylactosamines does not diminish the biological activity of pZP(B+C) preparations in competitive sperm-zona adhesion assays (20). Polylactosamines nonetheless partially occlude a peptide
epitope on pZPB (37) and attenuate binding affinity of pZP(B+C) toward
membrane vesicles (22). By analogy, in the native zona pellucida these
highly extended and intensely anionic glycans may modulate
(a) subunit interactions that drive assembly and maintain
stability of pZPB-pZPC complexes and (b) affinity of
pZPB-pZPC complexes toward zona-binding proteins on boar sperm.
Experiments performed with reconstituted pZPB-pZPC complexes in which one partner molecule bears a biotin label have permitted measurement of ligand binding affinity and confirmed obligate specificity of membrane-associated zona-binding proteins for pZPB-pZPC heteromers. Based on saturation binding curves (Fig. 6), binding affinities for (biotin·pZPB5)-pZPC complexes (EC50 = 4.3 nM) and pZPB5-(biotin·pZPC) complexes (EC50 = 1.7 nM) are comparable. The somewhat lower binding affinity of (biotin·pZPB5)-pZPC complexes may indicate an adverse affect of pZPB subunit biotinylation. Alternatively, the EC50 values may mirror the stoichiometry of pZPB and pZPC subunits in membrane-binding heterocomplexes, i.e. a pZPB:pZPC molar ratio of approximately 2-3:1. Additional data support this hypothesis. Titration experiments produced: (i) maximal receptor-binding activities at pZPB:pZPC molar ratios of 3.3:1 for biotin-pZPB5 + pZPC co-incubates and 1.4:1 for pZPB5 + biotin-pZPC co-incubates (Fig. 6), and (ii) optimal competitor activity at a pZPB:pZPC molar ratio of ~2:1 for pZPB5 + pZPC co-incubates (Fig. 2B). At present, more direct attempts to define the minimal size and subunit composition of heteromers capable of engaging membrane-associated zona-binding proteins have been precluded by the avid propensity of co-incubated pZPB and pZPC glycoproteins to assemble into high mass heterocomplexes.
Work from other laboratories supports the hypothesis that pZPB and pZPC are intimately associated within the native zona pellucida (38-40). When particulate pig zonae are digested with boar sperm acrosin under conditions where both suprastructure and sperm binding activity are minimally affected, pZPA is selectively degraded and the corresponding Mr 90,000 band is no longer detected on SDS gels. Staining intensity of the Mr 55,000 band is not affected indicating that the pZPB and pZPC glycoproteins resist acrosin digestion in the intact zona. Consequently, protease-resistant pZPB-pZPC complexes likely form the main structural framework of the pig zona matrix, whereas pZPA serves as a cross-linking molecule.
Whether ZPB-ZPC interactions contribute to zona architecture in other species is as yet uncertain. In the mouse, protease-resistant filaments comprised of ZP2 and ZP3 glycoproteins are interconnected via ZP1 cross-links to form a three-dimensional matrix (41). Although further molecular details have not been reported, ZP1 must establish molecular contact with ZP2 and/or ZP3 at cross-linkage sites. As the mouse ZP1, ZP2, and ZP3 glycoproteins are encoded by the respective members of the ZPB (42), ZPA (6), and ZPC (5) gene families, specific interactions between ZPB and ZPC gene products, i.e. ZP1 and ZP3, may in theory contribute to assembly and stability of the mouse zona matrix.
Aside from a potential structural role within the pig zona pellucida, pZPB-pZPC complexes function as high affinity ligands for cognate zona-binding proteins present in isolated boar sperm membrane vesicles. As neither subunit binds independently, our data define a ligand distinct from others previously described in the sperm-zona literature. Most notably, the mouse ZPC gene product, ZP3, acts independently as a sperm-adhesive molecule and as a secretagogue for acrosomal exocytosis (11, 43, 44). Moreover, kinetic analyses of binding of 125I-labeled acid-solubilized mouse zonae pellucidae to glutaraldehyde-fixed mouse sperm suggest that ZP3 binds to receptor(s) as a high affinity (Kd = 0.72 nM), multivalent ligand (45). Thus the mouse and the pig employ contrasting molecular mechanisms for constructing high affinity sperm-binding sites, i.e. zona glycoproteins acting: (i) independently as multivalent ligands (mouse: ZP3) or (ii) co-dependently as hetero-oligomers (pig: pZPB and pZPC).
In the rabbit, two reports suggest participation of ZPB and ZPC gene products in sperm-zona interactions. First, a recombinant ZPB-encoded protein (BV55) binds to rabbit zonae and also elicits blocking antibodies (46). Second, cross-linking and immunoprecipitation experiments (47) show that when incubated with heat-solubilized rabbit zona pellucida, the recombinant rabbit sperm protein rSp17 binds macromolecular complexes containing both the R55 (i.e. rZPB) (8) and R45 (i.e. rZPC) (13) zona proteins. Whether rSp17 has an obligate binding specificity for R55-R45 heteromers is an intriguing question that remains unanswered.
The present study raises important questions regarding the mechanism by which association of pZPB and pZPC glycoproteins creates a high affinity ligand and whether one or both subunits in this complex directly engage cognate zona-binding proteins. In one scenario, a conformational change concomitant with hetero-oligomerization may expose a cryptic adhesion domain in one subunit. Assuming that (i) domains responsible for subunit interactions are conserved across species and (ii) sperm adhesive determinants are species-specific, then experiments that test binding of chimeric ZPB-ZPC complexes to boar sperm membrane vesicles may provide insight into specificity of corresponding zona-binding proteins. In this context, a preliminary experiment has shown that Chinese hamster ovary expressed recombinant human pZPC promotes binding of biotin-pZPB5 to boar sperm membrane vesicles.3 Alternatively, pig sperm-zona interactions may mimic the gonadotropin ligand-receptor model (48) and both pZPB and pZPC may participate directly in binding, e.g. by creation of a high affinity adhesion domain consisting of oligosaccharides and/or peptide residues contributed by both molecules.
Zona carbohydrates play an essential role in mammalian gamete interactions (49), and several reports in the literature (18, 19, 23, 36, 50) provide evidence that the pig system conforms with this paradigm. The current study, where we demonstrate that boar sperm membranes exhibit an obligate specificity for heteromeric pZPB-pZPC complexes, emphasizes that attempts to identify bioactive carbohydrate ligands in the solid phase system by use of chemically or enzymatically liberated pig zona glycans are likely to prove futile. Alternative approaches, such as assembly of pZPB-pZPC complexes using either native pig zona proteins pretreated with glycosidases or recombinant pig zona proteins with foreign N- and O-glycans or ablated glycosylation sequons, are therefore required. By analogy to the gonadotropins (51), data from such experiments must be interpreted with caution as molecular modifications that alter the interaction between pZPB and pZPC subunits may in theory also diminish the avidity of reconstituted pZPB-pZPC complexes for cognate zona-binding proteins.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HD23163 (to E. C. Y.). Matrix assisted laser desorption/ionization mass spectrometry analysis was performed at the Michigan State University Mass Spectrometry Facility, which is supported in part by National Institutes of Health Grant RR00480 (to D. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Wayne State University, Dept. of Ob/Gyn, 275 East Hancock, Detroit, MI 48201. Tel.: 313-577-1228; Fax: 313-577-8554; E-mail: e.yurewicz{at}wayne.edu.
1 The abbreviations used are: HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; TBS, Tris-buffered saline; BSA, bovine serum albumin; MES, 2-(N-morpholino)ethane sulfonic acid.
2 E. C. Yurewicz, unpublished results.
3 E. C. Yurewicz and G. K. Fontenot, unpublished results.
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REFERENCES |
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