Purified and refolded recombinant bonnet monkey (macaca radiata) zona pellucida glycoprotein-B expressed in Escherichia coli binds to spermatozoa

Bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B (bmZPB), excluding the N-terminal signal sequence and the C-terminus transmembrane-like domain, has been expressed in Escherichia coli as polyhistidine fusion protein. A requirement of 4 M urea to maintain the purified protein in soluble state rendered it unsuitable for biological studies. Purification of refolded r-bmZPB without urea and devoid of lower molecular weight fragments was achieved by following an alternate methodology that involved purification of inclusion bodies to homogeneity and solubilization in the presence of a low concentration of chaotropic agent (2 M urea) and high pH (pH 12). The solubilized protein was refolded in the presence of oxidized and reduced glutathione. The circular dichroism spectra revealed the presence of both a helical and β sheet components in the secondary structure of the refolded r-bmZPB. The binding of the refolded r-bmZPB to the spermatozoa was evaluated by an indirect immunofluorescence assay and also by direct binding of the biotinylated r-bmZPB. The binding was restricted to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. In the acrosome-reacted spermatozoa a shift in the binding pattern of r-bmZPB was observed and it bound to the equatorial segment, postacrosomal domain, and midpiece region. Binding of biotinylated r-bmZPB was inhibited by cold r-bmZPB as well as by monoclonal and polyclonal antibodies generated against r-bmZPB. These results suggest that nonglycosylated bmZPB binds to capacitated as well as acrosome-reacted spermatozoa in a nonhuman primate and may have a functional role during fertilization

In silico identification and characterization of the SNPs in the human ASTL gene and their probable role in female infertility

Ovastacin (ASTL), a zinc metalloprotease, is released from a fertilized egg during exocytosis of cortical granules which occurs minutes after the sperm and egg fuse. ASTL cleaves ZP2, one of the four primary glycoproteins of human zona pellucida, and this cleavage prevents polyspermy, causes zona pellucida hardening, and also protects the pre-implantation embryo. Any perturbation in the activity of ASTL can thus disturb this process and may lead to infertility without changing the gross morphology of the oocyte. A small amount of ASTL is also released by unfertilized oocytes but its catalytic activity is absent as it is bound by its inhibitor, Fetuin-B (FETUB). Pre-mature release of ASTL when FETUB is absent also causes infertility. To identify and understand the structural and functional effects of deleterious SNPs of ASTL on its interaction with ZP2 and FETUB and hence on fertility, a total of 4,748 SNPs from the dbSNP database were evaluated using a variety of in silico tools. All of the 40 shortlisted nsSNPs were present in the catalytic domain of the protein. Comparison of the wild type with mutants using MutPred2 suggests an alteration in the catalytic activity/zinc binding site in many SNPs. Docking studies show the involvement of hydrophobic interactions and H bonding between ASTL and ZP2 and also between ASTL and FETUB. Four positions in ASTL involved in the hydrophobic interactions (P105 and D200 between ASTL and ZP2; D198 and L278 between ASTL and FETUB) and 5 in H bonding (E75 and R159 between ASTL and ZP2; and K93, R159, and C281 between ASTL and FETUB) have SNP’s associated with them validating their importance. Interestingly, a cluster of multiple SNPs was found in the motif 198DRD200, which is also a well-conserved region among several species. Statistical Coupling Analysis (SCA) suggested that the deleterious SNPs were present in the functionally important amino acid positions of ASTL and are evolutionarily coupled. Thus, these results attempt to identify the regions in ASTL, mutations in which can affect its binding with ZP2 or FETUB and cause female infertility

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<SUB>(223-348)</SUB>] 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 ~25 kDa. Lectin-binding studies documented the presence of both O- as well as N-linked glycans. The biotinylated r-bmZP3<SUB>(223-348)</SUB> 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<SUB>(223-348)</SUB> and baculovirus expressed r-bmZP3, devoid of signal sequence and transmembrane-like domain [r-bmZP3<SUB>(23-348)</SUB>] competitively inhibit its binding to the capacitated spermatozoa. Interestingly, binding of biotinylated r-bmZP3<SUB>(23-348)</SUB> to the capacitated sperm is also inhibited by nonbiotinylated r-bmZP3(223-348). In contrast to r-bmZP3<SUB>(23-348)</SUB>, r-bmZP3<SUB>(223-348)</SUB> 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

Primate recombinant zona pellucida proteins expressed in Escherichia coli bind to spermatozoa

To delineate the role of individual zona pellucida (ZP) glycoproteins during sperm–oocyte interaction, bonnet monkey (bm; Macaca radiata) ZPA (bmZPA), ZPB (bmZPB), and ZPC (bmZPC) have been cloned without native signal sequence and transmembrane-like domain, and expressed in Escherichia coli. Recombinant proteins have been purified from the inclusion bodies in presence of low concentration of chaotropic agent (2 M urea) and high pH (pH 12), and subsequently refolded in presence of oxidized and reduced glutathione. Binding of the recombinant refolded zona proteins to bonnet monkey spermatozoa in an indirect immunofluorescence assay revealed that recombinant bmZPC binds to the head region of the capacitated spermatozoa but does not bind to the acrosome reacted spermatozoa. Recombinant bmZPB binds to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. After induction of acrosome reaction by calcium ionophore A23187, the binding of recombinant bmZPB shifts to the equator, post-acrosome and midpiece of the spermatozoa. bmZPA binds to the principal segment of capacitated spermatozoa but the binding shifts to the equatorial segment, tip of the inner acrosomal membrane and midpiece in acrosome reacted spermatozoa. These studies suggest that polypeptide backbone is sufficient for the binding of ZPA, ZPB and ZPC to spermatozoa in non-human primates. Further studies with recombinant glycosylated zona proteins will help in delineating the role of carbohydrate moieties for higher affinity binding of the ligand to spermatozoa and subsequent signal transduction pathways

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 &amp; 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. Mol. Reprod. Dev. 70: 247-254, 2005

The Lectin Domain of the Polypeptide GalNAc Transferase Family of Glycosyltransferases (ppGalNAc Ts) Acts as a Switch Directing Glycopeptide Substrate Glycosylation in an N- or C-terminal Direction, Further Controlling Mucin Type O-Glycosylation

Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain in ppGalNAc T glycopeptide substrate utilization, we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N or C terminus of the glycopeptide substrate. Our results reveal that the presence and N- or C-terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled