DEVELOPMENTAL
BIOLOGY
Identification
154,309-317 (19%)
of a Region of Mouse Zona Pellucida Glycoprotein That Possesses Sperm Receptor Activity
mZP3
THOMAS K. ROSIERE’ AND PAUL M. WASSARMAN~ Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research, Center, Nutley, New Jersey 07110 Accepted July 16, 1992 The ability of mouse zona pellucida glycoprotein ZP3 (mZP.3) to function as a sperm receptor is attributable to certain of its oligosaccharides, not to its polypeptide (P. M. Wassarman, 1990. Development 10&l-17). Here, purified, radioiodinated mZP3 was digested by either papain or V8 protease, and the glycopeptides produced were fractionated by HPLC and assayed for sperm receptor activity in vitro. Each proteolytic digest of mZP3 contained a heavily glycosylated peptide, -55,000 apparent M,, that exhibited sperm receptor activity invitro. To determine the region of mZP3 polypeptide from which the active glycopeptides were derived, Western gel immunoblotting, employing an antiserum directed against a specific mZP3 peptide epitope, and automated amino-terminal amino acid sequencing were employed. Results of these experiments strongly suggest that the active glycopeptides produced by digestion of mZP3 with either papain or V8 protease are derived from the same region of the carboxy-terminal half of the mZP3 polypeptide. These and other findings are discussed in terms of mZP3 structure and function. 0 1992 Academic press, IIIC. INTRODUCTION
The mammalian egg extracellular coat, or zona pellucida (ZP), plays several important roles during the process of fertilization (Gwatkin, 1977; Wassarman, 1987a,b, 1991a; Yanagimachi, 1988; Dietl, 1989). For example, mZP3, one of three glycoproteins that constitute the mouse egg ZP, serves as a sperm receptor during species-specific binding of sperm to ovulated eggs (Wassarman, 1987a,b, 1988, 1990). mZP3 is -83,000 M, and consists of a -44,000 M, polypeptide (402 amino acids), three or four complex-type, asparagine (N-)-linked oligosaccharides, and an undetermined number of serine/ threonine (O-)-linked oligosaccharides (Wassarman, 1988, 1989, 1990, 1991b). A specific class of mZP3 Olinked oligosaccharides (-3900 M,) is responsible for the glycoprotein’s sperm receptor activity (SRA, Wassarman, 1988, 1989, 1990, 1991b). Thus, the initial species-specific binding of sperm to eggs during mammalian fertilization is a carbohydrate-mediated event. Previously, we reported that small glycopeptides present in Pronase digests of purified egg mZP3 exhibited SRA in vitro (Florman et aZ., 1984; Wassarman et ah, 1985). Here, we isolated and characterized mZP3 glycopeptides, produced by digestion of purified glycoprotein with either papain or V8 protease, in order to determine which region(s) of mZP3 possesses SRA. Results of these experiments suggest that such a region is located 1 Present address: Abbott Laboratories, One Abbott Park Road, Abbott Park, IL 60064-3500. ’ To whom correspondence should be addressed. 309
in the carboxy-terminal half of the mZP3 polypeptide. Evidence is also presented to suggest that this region of the mZP3 polypeptide is heavily glycosylated and, consequently, is a relatively protease-resistant portion of mZP3. A preliminary report of these results has appeared (Rosiere and Wassarman, 1990). MATERIALS
AND METHODS
Collection and culture of gametes and embryos. Sperm, ovulated eggs, and fertilized eggs were obtained from randomly bred, Swiss albino mice (CD-l; Charles River Breeding Laboratories) and cultured in vitro, as previously described (Bleil and Wassarman, 1980; Florman and Wassarman, 1985; Moller et aZ., 1990). Fertilized eggs were cultured for about 24 hr to obtain two-cell embryos. Purification and radiolabeling of mZP3. mZP3 was purified by HPLC from ZP that were isolated by Percoll (Pharmacia-LKB) gradient centrifugation of ovarian homogenates, as previously described (Bleil and Wassarman, 1986; Bleil et ah, 1988). Purified mZP3 was radioiodinated in 0.1 M sodium phosphate (pH 7.4)/0.1% SDS in the presence of carrier-free Na[1251](Amersham) and chloramine T (0.5 mg/ml) at room temperature for 15 min. The reaction was terminated with sodium bisulfite (1 mg/ml). Sufficient Na[‘251] was used such that all five mZP3 tyrosine residues were radiolabeled, to either monoiodo- or diiodotyrosine, and unincorporated Na[1251]was removed by using a Bio-Gel P-2 Bio-Spin column (Bio-Rad). Enzymatic digestion of putified mZP3. Purified mZP3 0012-1606/92 $5.00 Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
DEVELOPMENTALBIOLOGY
310
was digested by immobilized papain (Pierce) in 50 mM Hepes (pH 6.1)/10 mM EDTA/ZO mM cysteine/O.l% SDS, at an enzyme to mZP3 ratio of 0.5 to 1, at 37°C for 2 hr. Enzyme was separated from mZP3 glycopeptides by using a Bio-Gel P-2 Bio-Spin column in 0.2 M sodium phosphate (pH 6.6)/0.1% SDS, glycopeptides were fractionated by HPLC (Bio-Sil SEC-250 column, 300 x 7.8 mm [Bio-Rad]; flow rate, 0.1 ml/min) in the same buffer, and fractions were analyzed by gamma counting and SDS-PAGE. Alternatively, purified mZP3 was digested by V8 protease from Staphylococcus aureus (Sigma) in 50 mM phosphate buffer (pH 7.8)/0.1% SDS, at an enzyme to mZP3 ratio of at least 2 to 1, at 37°C for 8-16 hr. Samples were heated at 95°C for 5 min, and mZP3 glycopeptides were purified and analyzed as described above. In some cases, N-linked oligosaccharides were removed from mZP3 and mZP3 glycopeptides by digestion with recombinant N-glycanase (Genzyme) at 37°C for 12-18 hr. In these experiments, samples in 50 mM sodium phosphate (pH 7.5)/25 mM EDTA/l% 2-mercaptoethanol/O.l% SDS were heated to 95°C for 5 min, cooled, and brought to 1.5% octyl glucoside prior to addition of enzyme.
Antibody
production
and Western gel immunoblotting.
An mZP3-specific rabbit antiserum (Ra-mZP3; Kinloch et al., 1991), directed against a KLH-conjugated synthetic peptide that corresponds to amino acids 306-321 of the mZP3 polypeptide, was used on Western gel immunoblots. Proteins were subjected to SDS-PAGE and then transferred to nitrocellulose, and membranes were incubated for 1 hr in 20 mM Tris-HCl (pH 7.5)/3% BSA/150 mM NaC1/0.2% Tween-20 (“blocking solution”; TBST) and then for 1 hr in the presence of RamZP3 (antiserum diluted 1:500 in TBST containing 1% BSA). Unbound antibody was removed by extensive washing, and membranes were then incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad) and developed in the presence of nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Amino acid sequencing. Proteolytic digestion products of ‘251-mZP3 were transferred individually to polyvinylidene difluoride (PVDF) membranes (Immobilon Transfer; Millipore) and subjected to amino acid sequencing with an Applied Biosystems Model 470A sequenator equipped with on-line PTH analysis, essentially according to procedures previously described (Matsudaira, 1987). Sperm receptor activity assays. SRA was assessedby using an in vitro “competition assay,” essentially as previously described (Bleil and Wassarman, 1980; Florman and Wassarman, 1985; Moller et ah, 1990). Purified mZP3 or mZP3 glycopeptides were incubated for 1 hr
vOLUMIZ154,19%
with capacitated sperm in a 50-~1 drop prior to addition of lo-15 ovulated eggs and 2-4 two-cell embryos. After 20-30 min, eggs and embryos were pipeted to remove loosely associated sperm and fixed in 4% glutaraldehyde-PBS/PVP (4 mg/ml), and the number of sperm bound per egg was determined by light microscopy. RESULTS
Experimental
Rationale
Previously, we reported that small (-1500-6000 apparent M,.) mZP3 glycopeptides, produced during extensive digestion of purified glycoprotein by insolubilized Pronase, as well as purified mZP3 O-linked oligosaccharides released by alkaline reduction of purified glycoprotein, exhibited SRA in vitro (Florman et al., 1984; Florman and Wassarman, 1985; Wassarman et ab, 1985; Bleil and Wassarman, 1988; Wassarman, 1989). The mZP3 primary structure includes 71 serine plus threonine residues (not including the 22-amino acid putative signal sequence at the amino-terminus), distributed throughout the 402-amino-acid polypeptide (Kinloch et al., 1988; Ringuette et aL, 1988), that are potential glycosylation sites. Here, we used two proteases, papain and V8 protease, to produce a limited number of mZP3 glycopeptides, HPLC purified and tested the glycopeptides for SRA in vitro, and located the positions of the glycopeptides along the mZP3 polypeptide. The latter was accomplished by using an antiserum directed against a specific mZP3 peptide epitope and by automated aminoterminal amino acid sequencing of purified mZP3 glycopeptides. It should be noted that all molecular weights reported here should be considered as “apparent molecular weights,” since it is well known that glycoproteins and glycopeptides migrate anomolously on SDS-PAGE due, in part, to a low charge-to-mass ratio for the molecules (Leach et al., 1980). It should also be noted that the designation of the mZP3 polypeptide amino acid residue number is based on the 402-amino acid mature polypeptide, not the 424-amino acid nascent polypeptide containing an amino-terminal signal sequence.
SDS-PAGE
and HPLC
of mZP3 Glycopeptides
SDS-PAGE and HPLC analyses of papain digests of 1261-mZP3 revealed the presence of four radiolabeled glycopeptides, -55,000, -26,000, -13,000, and
BIOLOGY
Identification
154,309-317 (19%)
of a Region of Mouse Zona Pellucida Glycoprotein That Possesses Sperm Receptor Activity
mZP3
THOMAS K. ROSIERE’ AND PAUL M. WASSARMAN~ Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research, Center, Nutley, New Jersey 07110 Accepted July 16, 1992 The ability of mouse zona pellucida glycoprotein ZP3 (mZP.3) to function as a sperm receptor is attributable to certain of its oligosaccharides, not to its polypeptide (P. M. Wassarman, 1990. Development 10&l-17). Here, purified, radioiodinated mZP3 was digested by either papain or V8 protease, and the glycopeptides produced were fractionated by HPLC and assayed for sperm receptor activity in vitro. Each proteolytic digest of mZP3 contained a heavily glycosylated peptide, -55,000 apparent M,, that exhibited sperm receptor activity invitro. To determine the region of mZP3 polypeptide from which the active glycopeptides were derived, Western gel immunoblotting, employing an antiserum directed against a specific mZP3 peptide epitope, and automated amino-terminal amino acid sequencing were employed. Results of these experiments strongly suggest that the active glycopeptides produced by digestion of mZP3 with either papain or V8 protease are derived from the same region of the carboxy-terminal half of the mZP3 polypeptide. These and other findings are discussed in terms of mZP3 structure and function. 0 1992 Academic press, IIIC. INTRODUCTION
The mammalian egg extracellular coat, or zona pellucida (ZP), plays several important roles during the process of fertilization (Gwatkin, 1977; Wassarman, 1987a,b, 1991a; Yanagimachi, 1988; Dietl, 1989). For example, mZP3, one of three glycoproteins that constitute the mouse egg ZP, serves as a sperm receptor during species-specific binding of sperm to ovulated eggs (Wassarman, 1987a,b, 1988, 1990). mZP3 is -83,000 M, and consists of a -44,000 M, polypeptide (402 amino acids), three or four complex-type, asparagine (N-)-linked oligosaccharides, and an undetermined number of serine/ threonine (O-)-linked oligosaccharides (Wassarman, 1988, 1989, 1990, 1991b). A specific class of mZP3 Olinked oligosaccharides (-3900 M,) is responsible for the glycoprotein’s sperm receptor activity (SRA, Wassarman, 1988, 1989, 1990, 1991b). Thus, the initial species-specific binding of sperm to eggs during mammalian fertilization is a carbohydrate-mediated event. Previously, we reported that small glycopeptides present in Pronase digests of purified egg mZP3 exhibited SRA in vitro (Florman et aZ., 1984; Wassarman et ah, 1985). Here, we isolated and characterized mZP3 glycopeptides, produced by digestion of purified glycoprotein with either papain or V8 protease, in order to determine which region(s) of mZP3 possesses SRA. Results of these experiments suggest that such a region is located 1 Present address: Abbott Laboratories, One Abbott Park Road, Abbott Park, IL 60064-3500. ’ To whom correspondence should be addressed. 309
in the carboxy-terminal half of the mZP3 polypeptide. Evidence is also presented to suggest that this region of the mZP3 polypeptide is heavily glycosylated and, consequently, is a relatively protease-resistant portion of mZP3. A preliminary report of these results has appeared (Rosiere and Wassarman, 1990). MATERIALS
AND METHODS
Collection and culture of gametes and embryos. Sperm, ovulated eggs, and fertilized eggs were obtained from randomly bred, Swiss albino mice (CD-l; Charles River Breeding Laboratories) and cultured in vitro, as previously described (Bleil and Wassarman, 1980; Florman and Wassarman, 1985; Moller et aZ., 1990). Fertilized eggs were cultured for about 24 hr to obtain two-cell embryos. Purification and radiolabeling of mZP3. mZP3 was purified by HPLC from ZP that were isolated by Percoll (Pharmacia-LKB) gradient centrifugation of ovarian homogenates, as previously described (Bleil and Wassarman, 1986; Bleil et ah, 1988). Purified mZP3 was radioiodinated in 0.1 M sodium phosphate (pH 7.4)/0.1% SDS in the presence of carrier-free Na[1251](Amersham) and chloramine T (0.5 mg/ml) at room temperature for 15 min. The reaction was terminated with sodium bisulfite (1 mg/ml). Sufficient Na[‘251] was used such that all five mZP3 tyrosine residues were radiolabeled, to either monoiodo- or diiodotyrosine, and unincorporated Na[1251]was removed by using a Bio-Gel P-2 Bio-Spin column (Bio-Rad). Enzymatic digestion of putified mZP3. Purified mZP3 0012-1606/92 $5.00 Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
DEVELOPMENTALBIOLOGY
310
was digested by immobilized papain (Pierce) in 50 mM Hepes (pH 6.1)/10 mM EDTA/ZO mM cysteine/O.l% SDS, at an enzyme to mZP3 ratio of 0.5 to 1, at 37°C for 2 hr. Enzyme was separated from mZP3 glycopeptides by using a Bio-Gel P-2 Bio-Spin column in 0.2 M sodium phosphate (pH 6.6)/0.1% SDS, glycopeptides were fractionated by HPLC (Bio-Sil SEC-250 column, 300 x 7.8 mm [Bio-Rad]; flow rate, 0.1 ml/min) in the same buffer, and fractions were analyzed by gamma counting and SDS-PAGE. Alternatively, purified mZP3 was digested by V8 protease from Staphylococcus aureus (Sigma) in 50 mM phosphate buffer (pH 7.8)/0.1% SDS, at an enzyme to mZP3 ratio of at least 2 to 1, at 37°C for 8-16 hr. Samples were heated at 95°C for 5 min, and mZP3 glycopeptides were purified and analyzed as described above. In some cases, N-linked oligosaccharides were removed from mZP3 and mZP3 glycopeptides by digestion with recombinant N-glycanase (Genzyme) at 37°C for 12-18 hr. In these experiments, samples in 50 mM sodium phosphate (pH 7.5)/25 mM EDTA/l% 2-mercaptoethanol/O.l% SDS were heated to 95°C for 5 min, cooled, and brought to 1.5% octyl glucoside prior to addition of enzyme.
Antibody
production
and Western gel immunoblotting.
An mZP3-specific rabbit antiserum (Ra-mZP3; Kinloch et al., 1991), directed against a KLH-conjugated synthetic peptide that corresponds to amino acids 306-321 of the mZP3 polypeptide, was used on Western gel immunoblots. Proteins were subjected to SDS-PAGE and then transferred to nitrocellulose, and membranes were incubated for 1 hr in 20 mM Tris-HCl (pH 7.5)/3% BSA/150 mM NaC1/0.2% Tween-20 (“blocking solution”; TBST) and then for 1 hr in the presence of RamZP3 (antiserum diluted 1:500 in TBST containing 1% BSA). Unbound antibody was removed by extensive washing, and membranes were then incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad) and developed in the presence of nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Amino acid sequencing. Proteolytic digestion products of ‘251-mZP3 were transferred individually to polyvinylidene difluoride (PVDF) membranes (Immobilon Transfer; Millipore) and subjected to amino acid sequencing with an Applied Biosystems Model 470A sequenator equipped with on-line PTH analysis, essentially according to procedures previously described (Matsudaira, 1987). Sperm receptor activity assays. SRA was assessedby using an in vitro “competition assay,” essentially as previously described (Bleil and Wassarman, 1980; Florman and Wassarman, 1985; Moller et ah, 1990). Purified mZP3 or mZP3 glycopeptides were incubated for 1 hr
vOLUMIZ154,19%
with capacitated sperm in a 50-~1 drop prior to addition of lo-15 ovulated eggs and 2-4 two-cell embryos. After 20-30 min, eggs and embryos were pipeted to remove loosely associated sperm and fixed in 4% glutaraldehyde-PBS/PVP (4 mg/ml), and the number of sperm bound per egg was determined by light microscopy. RESULTS
Experimental
Rationale
Previously, we reported that small (-1500-6000 apparent M,.) mZP3 glycopeptides, produced during extensive digestion of purified glycoprotein by insolubilized Pronase, as well as purified mZP3 O-linked oligosaccharides released by alkaline reduction of purified glycoprotein, exhibited SRA in vitro (Florman et al., 1984; Florman and Wassarman, 1985; Wassarman et ab, 1985; Bleil and Wassarman, 1988; Wassarman, 1989). The mZP3 primary structure includes 71 serine plus threonine residues (not including the 22-amino acid putative signal sequence at the amino-terminus), distributed throughout the 402-amino-acid polypeptide (Kinloch et al., 1988; Ringuette et aL, 1988), that are potential glycosylation sites. Here, we used two proteases, papain and V8 protease, to produce a limited number of mZP3 glycopeptides, HPLC purified and tested the glycopeptides for SRA in vitro, and located the positions of the glycopeptides along the mZP3 polypeptide. The latter was accomplished by using an antiserum directed against a specific mZP3 peptide epitope and by automated aminoterminal amino acid sequencing of purified mZP3 glycopeptides. It should be noted that all molecular weights reported here should be considered as “apparent molecular weights,” since it is well known that glycoproteins and glycopeptides migrate anomolously on SDS-PAGE due, in part, to a low charge-to-mass ratio for the molecules (Leach et al., 1980). It should also be noted that the designation of the mZP3 polypeptide amino acid residue number is based on the 402-amino acid mature polypeptide, not the 424-amino acid nascent polypeptide containing an amino-terminal signal sequence.
SDS-PAGE
and HPLC
of mZP3 Glycopeptides
SDS-PAGE and HPLC analyses of papain digests of 1261-mZP3 revealed the presence of four radiolabeled glycopeptides, -55,000, -26,000, -13,000, and
