Expression of bonnet monkey (Macaca radiata) zona pellucida-3 (ZP3) in a prokaryotic system and its immunogenicity

An internal fragment (978 bp) corresponding to the bonnet monkey (Macaca radiata) ZP3, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by PCR from a full-length cDNA clone. The amplified Bam HI and Sacl restricted fragment was cloned in frama downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector. Recombinant ZP3 (r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strains SG13009[pREP4] and BL-21(DE3). Immunoblot with a murine monoclonal antibody, MA-451 (raised against porcine ZP3β—a homologue of bonnet ZP3, and cross-reactive with bonnet zona pellucida) revealed a predominant band of 50 kDa besides degraded fragments. Optimum expression of r-ZP3 was observed at 0.5 mM IPTG. Antisera generated in monkeys against synthetic peptides from the N-(23–45 aa residues) and C-(300–322 and 324–347 aa residues) termini of the deduced bonnet monkey precursor ZP3 sequence reacted with the r-ZP3 protein in ELISA. The r-ZP3 expressed in SG13009[pREP4] was purified on Ni-NTA resin under denaturing conditions and conjugated with diphtheria toxoid (DT). Immunization of a female rabbit and six female bonnet monkeys with the r-ZP3-DT conjugate generated antibodies reactive with r-ZP3 in ELISA. Rabbit r-ZP3 antiserum reacted with porcine ZP3β and bonnet r-ZP3 but failed to react with porcine ZP3α in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The availability of r-ZP3 will further help in evaluating its efficacy for fertility regulation and understanding the autoimmune oophoritis associated with ZP3 immunization in nonhuman primates. Mol. Reprod. Dev. 47:140–147, 1997. © 1997 Wiley-Liss, Inc.     

Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of sperm-egg binding

To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136-147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136-147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136-147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans.     

Antibodies generated in response to plasmid DNA encoding zona pellucida glycoprotein-B inhibit in vitro human sperm-egg binding

To investigate the immunogenicity of plasmid DNA encoding bonnet monkey (Macaca radiata) zona pellucida (ZP) glycoprotein-B (bmZPB), the cDNA corresponding to bmZPB, excluding the N-terminal signal sequence and C-terminus transmembrane-like domain, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRbmZPB). In vitro transfection of COS-1, COS-7, CHO, HEK-293, and UM-449 mammalian cells with VRbmZPB plasmid DNA led to the expression of bmZPB. Expression of bmZPB in transfected cells was cytosolic. Flow cytometry analysis of COS-1 cells transfected with VRbmZPB revealed that approximately 15% cells expressed bmZPB. The expressed bmZPB has an apparent molecular weight of 57 kDa. Immunization of male BALB/cJ mice with VRbmZPB plasmid DNA in saline as compared to VR1020 immunized group, elicited significant antibodies against E. coli expressed recombinant bmZPB as evaluated in ELISA. The antibodies generated by VRbmZPB plasmid DNA recognized bonnet monkey as well as human ZP. The immune sera obtained from mice immunized with VRbmZPB plasmid DNA also inhibited, in vitro, the binding of spermatozoa to the ZP in the hemizona assay. These studies, for the first time, demonstrate the feasibility of DNA vaccine to generate antibodies against ZP that recognize native protein and inhibit human sperm-oocyte binding.     

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.     

Cloning and expression of a cDNA encoding mouse kidney -amino acid oxidase

A cDNA encoding -amino acid oxidase (DAO;EC 1.4.3.3) has been isolated from a BALB/c mouse kidney cDNA library by hybridization with the cDNA for the porcine enzyme. Analysis of the nucleotide (nt) sequence of the clone revealed that it has a 1647-nt sequence with a 5′-terminal untranslated region of 68 nt that encodes 345 amino acids (aa), and a 3′-terminal untranslated region of 544 nt that contains the polyadenylation signal sequence ATTAAA. The deduced aa sequence showed 77 and 78% aa identity with the porcine and human enzymes, respectively. Two catalytically important aa residues, Tyr²²⁸ and His³⁰⁷, of the porcine enzyme, were both conserved in these three species. RNA blot hybridization analysis indicated that a DAO mRNA, of 2 kb, exists in mouse kidney and brain, but not liver. Synthesis of a functional mouse enzyme in Escherichia coli was achieved through the use of a vector constructed to insert the coding sequence of the mouse DAO cDNA downstream from the tac promoter of plasmid pKK223-3, which was designed so as to contain the lac repressor gene inducible by isopropyl-β- -thiogalactopyranoside. Immunoblot analysis confirmed the synthesis and induction of the mouse DAO protein, and the molecular size of the recombinant mouse DAO was found to be identical to that of the mouse kidney enzyme. Moreover, the maximum activity of the mouse recombinant DAO was estimated to be comparable with that of the porcine DAO synthesized in E. coli cells.     

Glycosylation of zona pellucida glycoprotein-3 is required for inducing acrosomal exocytosis in the bonnet monkey.

To investigate the role of polypeptide backbone vis-à-vis glycosylation of the putative primary sperm receptor, the bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 (bmZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was expressed as polyhistidine fusion protein either in Escherichia coli orusing baculovirus expression system. The recombinant bmZP3 (r-bmZP3) was purified using nickel-nitrilotriacetic acid resin and subsequently refolded in the presence of oxidized and reduced glutathione. SDS-PAGE and Western blot analysis revealed approximately 43 and approximately 52 kDa bands corresponding to E. coli and baculovirus expressed r-bmZP3, respectively. The r-bmZP3 purified from both E. coli and baculovirus binds to the principal segment of the acrosomal cap of the capacitated bonnet monkey spermatozoa as evaluated by indirect immunofluoresence assay. In a competitive inhibition assay, the binding of biotinylated baculovirus expressed r-bmZP3 to capacitated spermatozoa was inhibited not only by cold baculovirus expressed r-bmZP3 but also by E. coli expressed r-bmZP3 and vice-versa. Lectin binding studies revealed that the baculovirus r-bmZP3 has N-linked glycosylation with galactose and mannose residues. Capacitated spermatozoa, in the presence of baculovirus expressed r-bZP3, undergoes a significant increase (p < 0.01) in the acrosomal exocytosis as compared to control whereas E. coli expressed r-bmZP3 failed to have a significant effect. These results suggest that though the polypeptide backbone of ZP3 is sufficient for its binding to capacitated spermatozoa, yet glycosylation is required for induction of acrosomal exocytosis.     

Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding

To minimize ovarian dysfunction subsequent to immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing the antigenic B cell epitopes as immunogens have been proposed. In this study, attempts have been made to clone and express a recombinant chimeric protein encompassing the epitopes corresponding to bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1, amino acid residues 132-147), ZP glycoprotein-2 (bmZP2, amino acid residues 86-113), and ZP glycoprotein-3 (bmZP3, amino acid residues 324-347). The above chimeric recombinant protein (r-bmZP123) was expressed as a polyhistidine fusion protein in Escherichia coli. Immunoblot with murine monoclonal antibody, MA-813, generated against recombinant bmZP1 revealed a major band of approximately 10 kDa. The r-bmZP123 was purified on nickel-nitrilotriacetic acid resin under denaturing conditions. The female rabbits immunized with purified r-bmZP123 conjugated to diphtheria toxoid (DT) generated antibodies that reacted with r-bmZP123 and DT in an ELISA. In addition, the immune sera also reacted with E. coli expressed recombinant bmZP1, bmZP2, and bmZP3. In an indirect immunofluorescence assay, the antibodies against r-bmZP123 recognized native ZP of bonnet monkey as well as human. The immune sera also inhibited, in vitro, the binding of human spermatozoa to the human zona in the hemizona assay (HZA). These studies, for the first time, demonstrate the feasibility of assembling multiple epitopes of different ZP glycoproteins as a recombinant protein that elicit antibodies which are reactive with native zona and also inhibit, in vitro, human sperm-oocyte binding.     

Molecular cloning and sequencing of cDNA encoding for a novel testis-specific antigen

cDNA encoding for a sperm antigen, designated NZ-1, was cloned and sequenced from murine testis cDNA-λgt11 expression library using antibodies to human sperm surface antigens belonging to 14–18 kD molecular region. These sperm antigens are involved in zona pellucida binding and have tyrosine phyosphorylation activity. Computer generated translation analysis of 1395-bp cDNA yielded an open reading frame (ORF) of 152 aa with first ATG, Met start codon at nt 32 and the stop codon TGA at nt 487. The translated protein has a calculated molecular weight of 17.9 kD and a potential tyrosine phosphorylation site at aa 46–54, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced aa sequence indicated it to be a membrane-anchored peptide with a hydrophobic NH₂-terminus that is characteristic of a signal peptide. Extensive computer search in the GenBank, NBRF, and Swiss sequence banks, indicating it to be a novel protein. Northern blot analysis indicated testis-specific expression of NZ-1 antigen. The NZ-1 cDNA was subcloned into pGEX-1λT vector and expressed in glutathione-S-transferase gene fusion system to obtain the recombinant protein. The recombinant protein specifically reacted with the original antibodies raised against the native 14–18 kD sperm proteins. These findings suggest that the sperm-specific recombinant NZ-1 may find applications in the development of a contraceptive vaccine, and in studying the normal and abnormal sperm function and the signal transduction mechanism. Mol. Reprod. Dev. 48:449–457, 1997. © 1997 Wiley-Liss, Inc.     

Cloning and sequencing of cDNA encoding for a human sperm antigen involved in fertilization

A cDNA encoding for a sperm antigen, designated NZ-2, was cloned and sequenced from human testis cDNA-λgt11 expression library by using antibodies to human sperm surface antigens belonging to 14–18 kD molecular regions. These sperm antigens are involved in binding to zona pellucida of the human oocyte. Computer generated translation analysis of 963-bp cDNA yielded an open reading frame (ORF) of 163 amino acids (aa) with first ATG, Met start codon at nucleotide (nt) 335 and the stop codon TAA at nt 824. The NZ-2 cDNA has 335-bp 5′ and 139-bp 3′ noncoding regions. The translated protein has a calculated molecular weight of ∼19 kD, and has two casein kinase II (CK-2) sites at aa 94–97 and 149–152, respectively. Extensive computer search in the GenBank, National Biomedical Research Foundation (NBRF), and Swiss database indicates it to be a novel protein, having 99.5% nt sequence similarity, except for the first 40-bp, only with the human bacterial artificial chromosome (BAC) containing cloned human sperm DNA, at position 76935–76009. The in vitro translated product of T3 RNA polymerase by using NZ-2 cDNA digested with XhoI yielded a protein band of ∼20 kD, indicating it to be sense strand. The in vitro translated product of T7 RNA polymerase by using NZ-2 cDNA digested with NotI did not yield any protein band, indicating it to be antisense strand. The ∼20 kD protein was recognized specifically by the antisperm IgG, not by the control IgG in the Western blot procedure. Neither antisperm IgG nor control IgG recognized any protein band in the in vitro translation products of the antisense strand. The human genomic DNAs from three different cells/tissues namely, sperm, kidney, and testis when cut by HindIII, and then hybridized with the NZ-2 cDNA probe in the Southern blot procedure, showed restriction fragment length polymorphism (RFLP). The recombinant human sperm NZ-2 antigen may find applications in the development of a contraceptive vaccine, and diagnosis and treatment of infertility in humans. Mol. Reprod. Dev. 51:176–183, 1998. © 1998 Wiley-Liss, Inc.     

Baculovirus expressed C-terminal fragment of bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 inhibits ZP3-mediated induction of acrosomal exocytosis

Zona pellucida glycoprotein-3 (ZP3) has been postulated as the primary sperm receptor in various mammalian species including bonnet monkey (Macaca radiata). However, information on the domain responsible for its binding to spermatozoa is inadequate. In the present study, bonnet monkey ZP3 (bmZP3), corresponding to amino acid (aa) residues 223-348 [bmZP3(223-348)] has been cloned and expressed using baculovirus expression system. SDS-PAGE and Western blot analysis of the purified renatured recombinant protein revealed it as a closely spaced doublet of approximately 25 kDa. Lectin-binding studies documented the presence of both O- as well as N-linked glycans. The biotinylated r-bmZP3(223-348) binds to the acrosomal region of the capacitated spermatozoa but fails to bind to the acrosome-reacted spermatozoa as investigated by immunofluorescence studies. In ELISA, nonbiotinylated r-bmZP3(223-348) and baculovirus expressed r-bmZP3, devoid of signal sequence and transmembrane-like domain [r-bmZP3(23-348)] competitively inhibit its binding to the capacitated spermatozoa. Interestingly, binding of biotinylated r-bmZP3(23-348) to the capacitated sperm is also inhibited by nonbiotinylated r-bmZP3(223-348). In contrast to r-bmZP3(23-348), r-bmZP3(223-348) failed to induce acrosomal exocytosis in the capacitated sperm. Interestingly, it competitively inhibits the acrosomal exocytosis induced by r-bmZP3(23-348). These studies, for the first time, identify a domain of ZP3 capable of binding to capacitated spermatozoa and inhibiting ZP3-mediated induction of acrosomal exocytosis furthering our understanding of mammalian fertilization.     

Cloning and expression of cynomolgus monkey and baboon zona pellucida proteins

Partial clones for the three cynomolgus monkey (Macaca fasicularis) zona pellucida genes (cmZPA, cmZPB, and cmZPC) have previously been isolated. These partial clones contained the sequences for the C-terminal portion of each rcmZP protein. To obtain full-length clones for each cmZP, a fresh cynomolgus monkey ovarian cDNA library was constructed. PCR methodology was employed to speed the isolation of full-length clones for each cmZP cDNA. The 3' primers were designed based on sequence information from the previously identified clones; the 5' primers were designed using the human ZP sequences. The PCR technique yielded full-length clones of cmZPA and cmZPC, but not of cmZPB. Therefore, a genomic clone of cmZPB was isolated and the sequence determined. The exon/intron structure is nearly identical to the human ZPB exon/intron structure. New PCR primers were designed based on the cynomolgus monkey ZPB genomic sequence, and a full-length cmZPB cDNA was obtained. The same primers that were used to generate the cmZPB were also used to generate a baboon (Papio cynocephalus) ZPB (bZPB) cDNA. As was done previously for the human zona pellucida (hZP) cDNAs, the cmZP, and bZPB cDNAs were transferred to shuttle vectors for transfection into Chinese Hamster Ovary (CHO) cells. Stable cell lines for producing each ZP protein were isolated. Each cell line secreted the desired recombinant zona pellucida (rZP) protein into the culture medium, and each protein was purified using an established protocol. In terms of size and purity, the purified recombinant cmZP (rcmZP) and rbZPB proteins resemble the rhZP proteins.     

Incorporation of mouse zona pellucida proteins into the envelope of Xenopus laevis oocytes

 All vertebrate eggs have extracellular matrices, referred to as the zona pellucida in Mus musculus and the vitelline envelope in Xenopus laevis. The mouse zona, composed of three sulfated glycoproteins (ZP1, ZP2, ZP3), is critical for fertilization and early development, and mice lacking a zona pellucida produce no live offspring. The primary structures of mouse ZP1 (623 amino acids), ZP2 (713 amino acids) and ZP3 (424 amino acids) have been deduced from full-length cDNAs, but posttranslational modifications result in mature zona proteins with molecular masses of 200–180 kDa, 140–120 kDa, and 83 kDa, respectively. The vitelline envelope forms a similar structure around Xenopus eggs and contains three glycoproteins that are structurally related (39–48% amino acid similarity) to the three mouse zona proteins. To investigate whether the structural semblances are sufficient to allow incorporation of the mouse zona proteins into the Xenopus vitelline envelope, capped synthetic mRNAs encoding ZP1, ZP2, and ZP3 proteins were injected into the cytoplasm of stage VI Xenopus oocytes. After 20 h of incubation the oocytes were harvested, and posttranslationally modified zona proteins were detected with monoclonal antibodies specific to mouse ZP1, ZP2, and ZP3. The oocytes were imaged with confocal microscopy to detect individual zona proteins in the extracellular matrix of the oocytes, and this localization was confirmed biochemically. Thus the mouse zona proteins appear to have been sufficiently conserved through 350 million years of evolution to be incorporated into the extracellular envelope surrounding Xenopus eggs. Received: 5 January 1999 / Accepted: 12 February 1999