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• W2079802547 abstract "Zonadhesin is a mosaic protein in sperm membrane fractions that binds directly and in a species-specific manner to the extracellular matrix (zona pellucida) of the oocyte. The active form of pig zonadhesin from capacitated, epididymal spermatozoa comprises two covalently associated polypeptide chains of Mr105,000 (p105) and Mr 45,000 (p45). Here we report detection and characterization of multiple zonadhesin isoforms in freshly ejaculated cells. Antibodies to the predicted von Willebrand D0-D1, D1, and D3 domains of pig zonadhesin recognized p105, p45, and additional Mr 60,000–90,000 polypeptides in particulate fractions of uncapacitated cells. Although the p105/45 form constituted a minority of all zonadhesin forms in sperm membrane fractions, it was the predominant form capable of binding to the pig zona pellucida. Zonadhesin-binding sites were distributed over the entire zona pellucida. Anion exchange chromatography resolved active, p105/45 zonadhesin from the p60–90 inactive forms. Without disulfide bond reduction some zonadhesin wasMr ≥300,000, includingMr 300,000 and 900,000 proteins comprising in part multimers of p105/45. The multimeric forms did not bind the zona pellucida as avidly as did the p105/45 monomer. Expressed D1 and D3 domain fragments containing the CG(L/V)CG sequence motif spontaneously formed multimers at −246 mV Ehin vitro. Double Cys → Ser mutants of the D1 fragment formed multimers with the same apparent kinetics as the wild type protein. Zonadhesin localized to the apical head of pig spermatozoa. We conclude that a heterogeneous combination of specific proteolysis and intermolecular disulfide bond formation in the sperm head generates multiple forms of zonadhesin with differing avidities for the zona pellucida. Zonadhesin is a mosaic protein in sperm membrane fractions that binds directly and in a species-specific manner to the extracellular matrix (zona pellucida) of the oocyte. The active form of pig zonadhesin from capacitated, epididymal spermatozoa comprises two covalently associated polypeptide chains of Mr105,000 (p105) and Mr 45,000 (p45). Here we report detection and characterization of multiple zonadhesin isoforms in freshly ejaculated cells. Antibodies to the predicted von Willebrand D0-D1, D1, and D3 domains of pig zonadhesin recognized p105, p45, and additional Mr 60,000–90,000 polypeptides in particulate fractions of uncapacitated cells. Although the p105/45 form constituted a minority of all zonadhesin forms in sperm membrane fractions, it was the predominant form capable of binding to the pig zona pellucida. Zonadhesin-binding sites were distributed over the entire zona pellucida. Anion exchange chromatography resolved active, p105/45 zonadhesin from the p60–90 inactive forms. Without disulfide bond reduction some zonadhesin wasMr ≥300,000, includingMr 300,000 and 900,000 proteins comprising in part multimers of p105/45. The multimeric forms did not bind the zona pellucida as avidly as did the p105/45 monomer. Expressed D1 and D3 domain fragments containing the CG(L/V)CG sequence motif spontaneously formed multimers at −246 mV Ehin vitro. Double Cys → Ser mutants of the D1 fragment formed multimers with the same apparent kinetics as the wild type protein. Zonadhesin localized to the apical head of pig spermatozoa. We conclude that a heterogeneous combination of specific proteolysis and intermolecular disulfide bond formation in the sperm head generates multiple forms of zonadhesin with differing avidities for the zona pellucida. zona pellucida von Willebrand D von Willebrand factor modified radioimmune precipitation assay solution 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid polyacrylamide gel electrophoresis glutathioneS-transferase phosphate-buffered saline glutathione oxidized glutathione dithiothreitol effective potential in solution Adhesion of mammalian spermatozoa to the zona pellucida (ZP)1 is a complex process mediated by binding of sperm proteins to complementary ligands in the ZP (1Yanagimachi R. Knobil E. Neill J.D. The Physiology of Reproduction. Raven Press, Ltd., New York1994: 189-317Google Scholar, 2Bi, M., Wassler, M. J., and Hardy, D. M. (2002) inFertilization (Hardy, D. M., ed) in press, Academic Press, San Diego.Google Scholar). The complexity of this process derives partly from cellular changes that occur during gamete interactions. Spermatozoa undergo physiological changes in the female reproductive tract that are required for fertilization and are collectively called capacitation (1Yanagimachi R. Knobil E. Neill J.D. The Physiology of Reproduction. Raven Press, Ltd., New York1994: 189-317Google Scholar,3Jaiswal, B. S., and Eisenbach, M. (2002) inFertilization (Hardy, D. M., ed) in press, Academic Press, San Diego.Google Scholar). Although the molecular basis of capacitation is only partly understood, in some if not all species avidity of sperm-ZP adhesion increases as capacitation progresses. After capacitation is completed, the membranes involved in initial adhesion events are lost from the sperm surface during the acrosome reaction, but adhesion is sustained by interaction of newly exposed structures with the ZP (1Yanagimachi R. Knobil E. Neill J.D. The Physiology of Reproduction. Raven Press, Ltd., New York1994: 189-317Google Scholar, 2Bi, M., Wassler, M. J., and Hardy, D. M. (2002) inFertilization (Hardy, D. M., ed) in press, Academic Press, San Diego.Google Scholar). Unique adhesion molecule pairs likely function at different times during fertilization, and the activities of these molecules may change as fertilization progresses (2Bi, M., Wassler, M. J., and Hardy, D. M. (2002) inFertilization (Hardy, D. M., ed) in press, Academic Press, San Diego.Google Scholar). It is therefore important to assess the biochemical and functional properties of sperm adhesion molecules at each stage in the fertilization process. Several sperm proteins that may mediate adhesion to the ZP have been identified and characterized (2Bi, M., Wassler, M. J., and Hardy, D. M. (2002) inFertilization (Hardy, D. M., ed) in press, Academic Press, San Diego.Google Scholar). Among these molecules zonadhesin is unique in its ability to bind directly and in a species-specific manner to native, particulate ZP (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar, 5Hardy D.M. Garbers D.L. J. Biol. Chem. 1995; 270: 26025-26028Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Zonadhesin from pig (5Hardy D.M. Garbers D.L. J. Biol. Chem. 1995; 270: 26025-26028Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), mouse (6Gao Z. Garbers D.L. J. Biol. Chem. 1998; 273: 3415-3421Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), rabbit, 2I. A. Lea, P. Sivashanmugam, R. T. Richardson, and M. G. O'Rand, unpublished; GenBankTM accession number AF244982. and human (7Wilson M.D. Riemer C. Martindale D.W. Schnupf P. Boright A.P. Cheung T.L. Hardy D.M. Schwartz S. Scherer S.W. Tsui L.C. Miller W. Koop B.F. Nucleic Acids Res. 2001; 29: 1352-1365Crossref PubMed Scopus (45) Google Scholar) 3T. L. Cheung, M. J. Wassler, G. A. Cornwall, and D. M. Hardy, manuscript in preparation. spermatozoa is a mosaic protein with a predicted Type I integral membrane topology. In each of these species, the large extracellular region of the protein comprises primarily three domain types (meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin, and von Willebrand D (VWD)) that are present in other adhesion molecules (8Beckmann G. Bork P. Trends Biochem. Sci. 1993; 18: 40-41Abstract Full Text PDF PubMed Scopus (135) Google Scholar, 9Varki A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7390-7397Crossref PubMed Scopus (953) Google Scholar, 10Wagner D.D. Ann. Rev. Cell Biol. 1990; 6: 217-246Crossref PubMed Google Scholar). Although the domain structures of zonadhesin from these four mammals have been predicted from cDNA sequences, relatively little is known about the biochemical and functional properties of the proteins. The active form of pig zonadhesin in membrane fractions of capacitated, epididymal spermatozoa is a two-chain molecule with disulfide-bondedMr 105,000 and 45,000 polypeptides, both of which are derived from a predicted 2467-amino acid nascent precursor (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar, 5Hardy D.M. Garbers D.L. J. Biol. Chem. 1995; 270: 26025-26028Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). High Mr forms of zonadhesin have also been observed, suggesting the possible formation of covalent oligomers (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar). This possibility was further implied by the presence in the pig zonadhesin D1, D2, and D3 domains of a conserved CG(L/V)CG sequence motif (5Hardy D.M. Garbers D.L. J. Biol. Chem. 1995; 270: 26025-26028Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) that is important for the oligomerization and proper function of von Willebrand factor (11Mayadas T.N. Wagner D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3531-3535Crossref PubMed Scopus (176) Google Scholar) and for the oligomerization of porcine submaxillary mucin (12Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 34527-34534Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 13Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 6982-6988Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Perez-Vilar J. Hill R.L. J. Biol. Chem. 1999; 274: 31751-31754Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). These observations suggested that the protein at a minimum undergoes limited proteolysis and possibly also oligomerization as occurs in the functional maturation of vWF and other D-domain proteins (10Wagner D.D. Ann. Rev. Cell Biol. 1990; 6: 217-246Crossref PubMed Google Scholar). However, it is unclear when during sperm maturation such post-translational processing occurs or whether it is important for the ZP binding activity of zonadhesin. Here we report that heterogeneous post-translational processing gives rise to multiple isoforms of pig zonadhesin in freshly ejaculated spermatozoa. Among these, only forms comprising the p105 and p45 polypeptides possess ZP binding activity, and the monomeric p105/45 form binds more avidly than do higher order covalent oligomers. Furthermore, we find that zonadhesin binds uniformly to homologous ZP and localizes to the apical head of pig spermatozoa. These properties further support a function for zonadhesin in sperm adhesion to the extracellular matrix of the egg. Boar spermatozoa in extended, freshly ejaculated semen were washed and immediately disrupted by N2 cavitation at 650 p.s.i. (15Haden N.P. Hickox J.R. Scott W.C. Hardy D.M. Biol. Reprod. 2000; 63: 1839-1847Crossref PubMed Scopus (15) Google Scholar). Particulate fractions enriched in sperm plasma membranes were isolated from suspensions of disrupted cells by differential centrifugation (15Haden N.P. Hickox J.R. Scott W.C. Hardy D.M. Biol. Reprod. 2000; 63: 1839-1847Crossref PubMed Scopus (15) Google Scholar) as for previous studies with cauda epididymal spermatozoa (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar, 5Hardy D.M. Garbers D.L. J. Biol. Chem. 1995; 270: 26025-26028Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Solutions for sperm fractionations were buffered at pH 7.5 and contained EDTA (1 mm) and diisopropyl fluorophosphate (1 mm) to prevent proteolysis by acidic proteases, Ca2+-dependent metalloproteinases, or serine proteases, respectively. In some experiments, solutions also contained 1 mm iodoacetamide to inhibit thiol proteases and to prevent thiol oxidation. Isolated membrane fractions in 20 mm NaHEPES, 130 mm NaCl, 1 mm EDTA, pH 7.5 (HNE), were stored at −70 °C. Porcine ZP were isolated from sliced ovaries by stepwise sieving through screens (16Dunbar B.S. Wardrip N.J. Hedrick J.L. Biochemistry. 1980; 2: 356-365Crossref Scopus (187) Google Scholar) and then further purified by ultracentrifugation through Percoll (Amersham Pharmacia Biotech) gradients (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar). Isolated ZP in HNE were stored at −70 °C. Detergent-solubilized proteins from sperm membrane fractions were mixed with isolated ZP, and zonadhesin that bound directly to the particulate, native ZP was detected either by Western blotting (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar, 5Hardy D.M. Garbers D.L. J. Biol. Chem. 1995; 270: 26025-26028Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) or by epifluorescence. For localization of binding sites, sperm proteins were biotinylated (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar) prior to solubilization and incubation with ZP. The ZP with bound sperm proteins were washed extensively with 20 mm NaHEPES, 0.5m NaCl, 1 mm EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% SDS, pH 7.5 (mRIPA) (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar), and then bound, biotinylated proteins were detected by incubating for 15 min at 22 °C with Texas Red-labeled streptavidin (Molecular Probes, Eugene, OR) diluted 10,000-fold in 10 mm Tris-HCl, 150 mm NaCl, 0.1% (v/v) Tween 20, pH 7.5 (TBST). After washing three times for 5 min with TBST, ZP were dropped on coverslips, air dried, mounted with Fluoromount G (Electron Microscopy Sciences, Fort Washington, PA), and viewed by epifluorescence. ZP-bound forms of zonadhesin were characterized by Western blotting. Biotinylated zonadhesin polypeptides that remained bound to ZP after washing with mRIPA were detected by probing blots with horseradish peroxidase-streptavidin (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar). Alternatively, zonadhesin polypeptides that remained bound after washing with 1% CHAPS/HNE were detected by probing blots with specific antisera as described below. The 1.7-kilobase EcoRI fragment of pig zonadhesin cDNA clone M2 (in pBluescript) was subcloned into the EcoRI site of pET-23d. The 5′ sticky end of this fragment came from theEcoRI site in the adapter used to construct the cDNA library (5Hardy D.M. Garbers D.L. J. Biol. Chem. 1995; 270: 26025-26028Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), and its 3′ sticky end came from the EcoRI site at nucleotides 4063–4068 of the zonadhesin composite cDNA (GenBankTM accession number U40024). This construct specified an Mr 64,000 fusion protein comprising 20 amino acids of N-terminal vector-encoded protein, 19 amino acids of C-terminal vector-encoded protein (including a hexahistidine tag), and zonadhesin amino acids Pro683–Ser1224. The fusion protein was expressed in Escherichia coli strain BL21/DE3 by induction with 0.5 mm isopropylthiogalactoside for 2 h at 37 °C and isolated from inclusion bodies by preparative SDS-PAGE and electroelution. Asp-Pro bonds of the purified D0-D1 fusion protein were hydrolyzed for 36 h with 70% formic acid at 37 °C. The final hydrolysates contained a mixture of proteins with Mr values corresponding to partial hydrolysis products predicted from the deduced amino acid sequence, including an Mr 33,000 core polypeptide. Hydrolysates were lyophilized to remove formic acid prior to injection. Two female New Zealand White rabbits were immunized (intramuscular) with 0.2–0.5 mg of protein each in 1 ml of Freund's complete adjuvant (day 0). Booster injections (intramuscular) on day 45 consisted of 0.2–0.5 mg of protein each in 1 ml of Freund's incomplete adjuvant. Antisera were recovered from blood obtained by terminal exsanguinations on day 58. Glutathione S-transferase (GST) fusion proteins comprising in part amino acids Ser923–Met993 of the D1 domain or amino acids Ile1684–Pro1788 of the D3 domain were expressed in E. coli strain BL21. Polymerase chain reaction products (D1 sense primer, 5′-AGTGGATCCAGCACCTTCTCTGG-3′; D1 antisense primer, 5′-ATAGAATTCTGCTAGGCCGTGTTG-3′; D3 sense primer, 5′-CATCGGATCCCAGGTCAAGTTTGACGG-3′; and D3 antisense primer, 5′-GGGGAATTCTAGGCCGCCTG-3′; underlined bases denote mismatches introduced to create restriction sites and stop codons) encoding the D1 and D3 domain segments were directionally cloned into the BamHI andEcoRI sites of pGEX-2T, and fusion protein expression was induced with 0.1 mm isopropylthiogalactoside at 37 °C for 2 h. After washing the bacteria with 10 mmNaPO4, 150 mm NaCl, pH 7.4 (PBS), soluble fusion proteins were extracted by sonicating cell pellets in PBS containing 0.5 mm diisopropyl fluorophosphate, 1.0 mm EDTA, 10 mm E64, and 0.2% Triton X-100. Cell lysates were applied to a glutathione (GSH)-Sepharose column (15 ml of bed volume) equilibrated at 22 °C in PBS. Nonbinding proteins were washed through with PBS, and fusion proteins were eluted with 5 mm GSH in 50 mm Tris-HCl, pH 8.0. Eluted fusion proteins were present at concentrations of 5–8 mg/ml in the pooled, peak fractions, and with prior disulfide bond reduction migrated as single bands in SDS-PAGE (10% gels). Total yields of purified fusion protein were 40–45 mg/500 ml of culture. Four female New Zealand White rabbits were immunized (intramuscular) with 1 mg of purified fusion protein/animal (two with GST-D1 and two with GST-D3) emulsified in 0.5 ml of Freund's complete adjuvant (day 0). Booster injections consisted of 1 mg of purified fusion protein/animal emulsified in 0.5 ml of Freund's incomplete adjuvant (intramuscular) on day 42, and 1 mg of soluble protein/animal in PBS (subcutaneous) on days 49 and 70. Antisera were recovered from blood obtained by terminal exsanguinations on day 81. Purified GST (100 mg), GST-D1 (70 mg), and GST-D3 (70 mg) were dialyzed at 4 °C for >16 h in 0.1 mNaHCO3, 0.5 m NaCl, pH 8.3, to remove GSH and exchange into conjugation buffer. Dialyzed proteins were each coupled at 10 mg/ml swelled gel to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech). After washing by suction on a glass filter to remove uncoupled proteins, the remaining activated groups were blocked with 1 m ethanolamine, and the conjugated resins were washed with three cycles of alternating pH (0.1 m acetate, 0.5 m NaCl, pH 4.0, and 0.1 mNaHCO3, 0.5 m NaCl, pH 8.3). The affinity matrices were poured into 1-cm-diameter glass columns, equilibrated in PBS containing 0.02% NaN3, and then stored at 4 °C. Antibodies to GST were removed by passing 20 ml of antisera through a 10-ml bed volume GST-Sepharose column equilibrated at 22 °C in PBS. Antibodies to zonadhesin D1 or D3 domains were then affinity purified from their anti-GST-depleted sera by chromatography on GST-D1 or GST-D3 columns, respectively (7 ml of bed volume each, equilibrated in PBS at 22 °C). Elution of bound antibodies with 0.2 m sodium citrate, 0.15 m NaCl, pH 3.0, was monitored continuously byA280. Peak fractions were pooled and immediately adjusted to pH 7 by addition of 1 m Tris (unbuffered). Antibodies to GST that were removed in the initial depletion steps were similarly eluted from GST-Sepharose and recovered for use as affinity-purified control antibodies. All purified antibodies were stored at −70 °C. 20 mg of affinity-purified antibody to the D3 domain (11.4 mg from rabbit R128 and 8.6 mg from rabbit R129) were desalted into 0.1 mNaHCO3, 0.5 m NaCl, pH 8.3 (coupling buffer) in two runs on four tandem 5-ml HiTrap desalting columns (Amersham Pharmacia Biotech). Desalted protein (15 mg in 8.8 ml) was coupled to 0.43 g (dry weight) of freshly swollen CNBr-activated Sepharose 4B. A protein assay of uncoupled protein confirmed that more than 95% of the antibody (>14.5 mg) was coupled to the affinity matrix (1.5 ml of packed volume), which after blocking and washing as for the fusion protein affinity matrices was equilibrated in PBS containing 0.02% NaN3 and stored at 4 °C. Sperm membrane fractions (100 mg protein) were Dounce homogenized in 10 ml of 1% SDS/HNE and incubated for 30 min at 22 °C. The homogenate was diluted to 100 ml with HNE containing 0.5 mm diisopropyl fluorophosphate, 0.56% (w/v) sodium deoxycholate, and 1.1% (v/v) hydrogenated Triton X-100 to produce the composition of mRIPA, a detergent solution in which zonadhesin retains its ZP binding activity (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar). After ultracentrifugation for 1 h at 100,000 × g at 2 °C, up to 50 ml of the supernatant solution (mRIPA extract) was applied to a 1.5-ml anti-D3 column, and nonbound proteins were washed through with mRIPA untilA280 (monitored continuously) returned to base line. The column was further washed with 10 ml of HNE containing 1% (v/v) hydrogenated Triton X-100, and then the protein was eluted with 10 ml of 0.2 m sodium citrate, 0.15 mNaCl, 1% (v/v) hydrogenated Triton X-100, pH 3.0. Eluted protein fractions containing purified zonadhesin were pooled, adjusted to pH 7 with 1 m Tris (unbuffered), and stored at −70 °C. Two female New Zealand White rabbits were immunized (intradermal) with 100 µg of purified zonadhesin holoprotein/animal emulsified in 1 ml of Freund's complete adjuvant (day 0). After boosting with 100 µg of purified protein/animal emulsified in 1.2 ml of Freund's incomplete adjuvant (intramuscular) on day 45, antisera were recovered from blood obtained by terminal exsanguinations on day 60. Mutations in the pGEX-2T construct encoding GST-D3 were introduced with T4 polymerase-based GeneEditor (Promega Corp., Madison, WI), and those in the construct encoding GST-D1 were introduced with polymerase chain reaction-based QuickChange (Stratagene Inc., La Jolla, CA) without modification of the vendor's instructions. The primer for generating the double Cys → Ser mutant of the 1709CGVCG1713 motif in GST-D3 (C1709S,C1712S) was 5′-ACGGAAGGACCTCCGGCGTGAGCGGGAACTTCA-3′ (altered bases in mutagenesis primer sequences are underlined). Primers for generating mutants of the933CGLCG937 motif in GST-D1 were: 5′-CTCTGGCAAACTCTCTGGTCTCTGTGGCG-3′ (C933S sense); 5′-CGCCACAGAGACCAGAGAGTTTGCCAGAG-3′ (C933S antisense); 5′-CTCTGTGGTCTCAGTGGCGACTATGACGG-3′ (C936S sense); 5′-CCGTCATAGTCGCCACTGAGACCACAGAG-3′ (C936S antisense); 5′-CTCTGGCAAACTCTCTGGTCTCAGTGGCGACTATG-3′ (C933S,C936S sense); and 5′-CATAGTCGCCACTGAGACCAGAGAGTTTGCCAGAG-3′ (C933S,C936S antisense). Mutated constructs were verified by double-stranded DNA sequencing using insert-flanking pGEX primers (sense: 5′-AATCGGATCTGGTTCCG-3′; antisense: 5′-CGTCAGTCAGTCACGAT-3′). Mutant fusion proteins were expressed in BL21 cells and purified by GSH affinity chromatography as for the wild type proteins. SDS-PAGE and Western blotting were done as described previously (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar, 18Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44923) Google Scholar, 19Hardy D.M. Wild G.C. Tung K.S. Biol. Reprod. 1987; 37: 189-199Crossref PubMed Scopus (29) Google Scholar, 20Morrissey J.H. Anal. Biochem. 1981; 117: 307-310Crossref PubMed Scopus (2940) Google Scholar). Two-dimensional SDS-PAGE was also done as described previously (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar), except that gradient gels (4–12% linear) were used for each dimension to resolve both high Mr zonadhesin isoforms and their constituent polypeptides. Dilutions used to detect zonadhesin on Western blots of pig sperm membranes were: D0-D1 antisera, 1:50,000; D1 antibody, 1:5,000; D3 antibody, 1:50,000; holoprotein antisera, 1:50,000, each in TBST. Bound antibody was detected with horseradish peroxidase-conjugated secondary antibody (BIOSOURCE International, Camarillo, CA) diluted 1:50,000 in TBST and development by chemiluminescence (SuperSignal; Pierce). Wild type and mutant fusion proteins were purified by GSH affinity chromatography in the presence of 1 mm DTT to stabilize the expressed proteins in their monomeric forms. The purified fusion proteins were then desalted on Sephadex G50 spin columns equilibrated in 50 mm Tris-HCl, pH 7.4, containing 1 mm DTT. For time course studies, oxidized glutathione (GSSG) was added to desalted fusion protein (1 µg in Tris-DTT) to a final concentration of 25 mm, which upon reaction with DTT produced a 24 mm GSSG/2 mm GSH redox buffer with an effective potential in solution (Eh) at pH 7.4 of −246 mV. The reactions were incubated at 37 °C, and disulfide bond formation was terminated at various times by adding iodoacetamide to 60 mm. To determine dependence of multimer formation onEh, 5–25 mm GSSG was added to reactions to produce redox buffers ranging from −269 to −246 mV Eh. After incubating for 2 h at 37 °C, the reactions were terminated with iodoacetamide as for time course studies. Multimers in the terminated reactions were separated by SDS-PAGE and detected by staining with Coomassie Blue. All steps for immunolocalization experiments were done at 22 °C. Spermatozoa were recovered from pig epididymides and washed with HEPES-buffered medium as described previously (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar). For immunofluorescence, the cells were smeared on coverslips, air dried, and then fixed in methanol for 30 min. After blocking 30 min with 10% (v/v) heat-inactivated goat serum in PBS, the coverslips were floated 1 h on D0-D1 antisera diluted 1:400 in PBS/heat-inactivated goat serum. After washing coverslips with PBS, bound antibody was detected by incubating for 1 h with Texas Red-conjugated antibody to rabbit immunoglobulin (BIOSOURCE International) diluted 1:400 in PBS/heat-inactivated goat serum. After a final wash with PBS, coverslips were mounted with Fluoromount G and viewed by epifluorescence and phase contrast microscopy. Zonadhesin from membrane fractions of capacitated, epididymal spermatozoa bound directly and with high affinity to intact ZP (Fig.1). The bound zonadhesin comprised p105 and p45 polypeptides (Fig. 1a) as previously observed (4Hardy D.M. Garbers D.L. J. Biol. Chem. 1994; 269: 19000-19004Abstract Full Text PDF PubMed Google Scholar). Although earlier work established the species specificity of this interaction, the distribution of zonadhesin-binding sites in the pig ZP has not been characterized. We therefore visualized ZP-bound zonadhesinin situ by affinity fluorescence (Fig. 1, b andc). Zonadhesin protein was detected on the entire ZP, indicating that its binding sites were not regionalized in the ZP structure. In addition, the relative evenness of the labeling suggested that the binding sites are intrinsic to the ZP and not associated with adherent materials from the cumulus cell matrix or other potential contaminants of the ZP preparation. The locations of p105 and p45 tryptic peptides in the sequence of the pig zonadhesin precursor indicated that p45 comprises in part the D1 domain and that p105 comprises in part the D2 and D3 domains (ref. (5Hardy D.M. Garbers D.L. J. Biol. Chem. 1995; 270: 26025-26028Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and Fig. 2). To detect zonadhesin isoforms in spermatozoa and to characterize their polypeptide compositions, we prepared various domain-directed antisera and affinity-purified antibodies. The deduced sequence of the precursor specified numerous potentially antigenic regions, including segments located in approximately the same positions within the D1 and D3 domains that exhibited high predicted hydrophilicity, surface probability, and flexibility (Fig. 2). Accordingly, we raised antisera to a Gene 10 fusion protein spanning the D0 and D1 domains (Pro683–Ser1224), as well as to two GST fusion proteins comprising in part the short, antigenic segments identified in the D1 and D3 domains (Ser923–Met993 and Ile1684–Pro1788, respectively; Figs. 2 and3). We also raised antisera to whole zonadhesin isolated from membrane fractions of pig spermatozoa (Fig.3).Figure 3Production and characterization of zonadhesin antisera and antibodies.a, preparation and specificity of antisera and antibodies to recombinant fusion proteins. Shown are protein stains and Western blots of SDS-PAGE (10% gels, protein disulfides reduced) as indicated. Purified, recombinant zonadhesin fusion proteins (lanes labeled G10-D0D1-H6 (where “H6” indicates a His6 tag), GST-D1, andGST-D3) each migrated primarily as single, Coomassie Blue-stained bands in overloaded gels. To generate the immunogen for production of antisera to the D0-D1 domains, the purified Gene 10 fusion protein spanning Pro683–Ser1224 of pig zonadhesin (lane labeled G10-D0D1-H6) was partially hydrolyzed at Asp-Pro bonds (lane labeledDP-hydrolyzed). The three predominant bands visible in the hydrolyzed preparation (asterisks) corresponded to Pro724–Asp1191 (51,500 Da; top band), a mixture of Pro724–Asp1107 and Pro807–Asp1191 (42,500 and 42,300 Da, respectively; middle band), and Pro807–Asp1107 (33,200 Da; bottom band). GST fusion proteins (lanes labeledGST-D1 and GST-D3) were used to prepare affinity-purified antibodies to segments spanning Ser923–Met993 of the pig zonadhesin D1 domain and Ile1684–Pro1788 of the D3 domain. Specificity of the antisera and antibodies was determined by Western blotting mixtures of the fusion proteins. Arrowheads mark the locations of the three fusion proteins in a mixture of 50 ng of each partially pure protein (lane labeled Silver stain). Note" @default.
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