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S K Gupta - One of the best experts on this subject based on the ideXlab platform.
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baculovirus expressed c terminal fragment of bonnet monkey Macaca Radiata zona pellucida glycoprotein 3 inhibits zp3 mediated induction of acrosomal exocytosis
Molecular Reproduction and Development, 2005Co-Authors: Gagandeep Kaur Gahlay, Deepika Batra, S K GuptaAbstract: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 ∼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. Mol. Reprod. Dev. 71: 237–244, 2005. © 2005 Wiley-Liss, Inc.
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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
Molecular Reproduction and Development, 2005Co-Authors: Neela Sivapurapu, Gagandeep Kaur Gahlay, Akiko Hasegawa, Koji Koyama, S K GuptaAbstract: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. Mol. Reprod. Dev. 70: 247–254, 2005. © 2005 Wiley-Liss, Inc.
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purified and refolded recombinant bonnet monkey Macaca Radiata zona pellucida glycoprotein b expressed in escherichia coli binds to spermatozoa
Biology of Reproduction, 2001Co-Authors: Chhabi K Govind, Gagandeep Kaur Gahlay, Sangeeta Choudhury, S K GuptaAbstract:Abstract 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 α 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 assa...
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immunocontraceptive potential of recombinant bonnet monkey Macaca Radiata zona pellucida glycoprotein c expressed in escherichia coli and its corresponding synthetic peptide
Reproductive Biomedicine Online, 2001Co-Authors: Renuka Kaul, Chhabi K Govind, Neela Sivapurapu, Abhijit Afzalpurkar, V Srikanth, S K GuptaAbstract:Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for development of immunocontraceptive vaccines. In this study, the efficacy to block fertility by immunization with recombinant bonnet monkey (Macaca Radiata) zona pellucida glycoprotein-C (r-bmZPC) expressed in Escherichia coli and its synthetic peptide (P4: KGDCGTPSHSRRQPHVVSQWSRSA, aa residues 324-347) conjugated to diphtheria toxoid (DT) has been evaluated in a homologous system. Female bonnet monkeys, immunized with P4-DT conjugate showed better immunocontraceptive potential as compared to an r-bmZPC-DT immunized group. In spite of high anti-P4 antibody titres, animals continued to have ovulatory cycles and showed no disturbance in cyclicity (except summer amenorrhoea). No ovarian pathology was observed in the P4 immunized group. These results suggest that immunization with the P4 may lead to block in fertility without obvious ovarian dysfunction. However, further inputs are required to identify additional ZP based B-cell epitopes to enhance the contraceptive efficacy.
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refolding structural transition and spermatozoa binding of recombinant bonnet monkey Macaca Radiata zona pellucida glycoprotein c expressed in escherichia coli
FEBS Journal, 2000Co-Authors: Ashok K Patra, Gagandeep Kaur Gahlay, S K Gupta, Vijaya B V Reddy, Amulya K PandaAbstract:An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca Radiata) zona pellucida glycoprotein-C (bmZPC), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned in pQE-30 vector and the protein expressed as inclusion bodies in Escherichia coli. Recombinant bmZPC (r-bmZPC) was solubilized from purified inclusion bodies in the absence of a high concentration of chaotropic agents and was subsequently refolded. Use of a low concentration of urea (2 m) during solubilization of r-bmZPC helped to minimize the extent of protein aggregation during refolding of the recombinant protein, and retain the existing native-like secondary structure that was essential for proper folding. Purified r-bmZPC appeared as a dominant band of 43 kDa on SDS/PAGE and Western blot. Although it lacked carbohydrate moieties, the purified and refolded r-bmZPC bound to the head region of bonnet monkey spermatozoa, confirming the existence of a native-like conformation. CD revealed a maximum at 200 nm and a single broad minimum extending from 209 to 216 nm, indicating the presence of both α-helical and β-sheet conformations in the refolded r-bmZPC. Two different phases of transition were observed by urea-gradient electrophoresis, suggesting the existence of multiple intermediate stages during the unfolding of r-bmZPC. The availability of refolded r-bmZPC will help in elucidating its role during the complex cascade of events during fertilization.
Gagandeep Kaur Gahlay - One of the best experts on this subject based on the ideXlab platform.
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baculovirus expressed c terminal fragment of bonnet monkey Macaca Radiata zona pellucida glycoprotein 3 inhibits zp3 mediated induction of acrosomal exocytosis
Molecular Reproduction and Development, 2005Co-Authors: Gagandeep Kaur Gahlay, Deepika Batra, S K GuptaAbstract: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 ∼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. Mol. Reprod. Dev. 71: 237–244, 2005. © 2005 Wiley-Liss, Inc.
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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
Molecular Reproduction and Development, 2005Co-Authors: Neela Sivapurapu, Gagandeep Kaur Gahlay, Akiko Hasegawa, Koji Koyama, S K GuptaAbstract: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. Mol. Reprod. Dev. 70: 247–254, 2005. © 2005 Wiley-Liss, Inc.
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purified and refolded recombinant bonnet monkey Macaca Radiata zona pellucida glycoprotein b expressed in escherichia coli binds to spermatozoa
Biology of Reproduction, 2001Co-Authors: Chhabi K Govind, Gagandeep Kaur Gahlay, Sangeeta Choudhury, S K GuptaAbstract:Abstract 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 α 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 assa...
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refolding structural transition and spermatozoa binding of recombinant bonnet monkey Macaca Radiata zona pellucida glycoprotein c expressed in escherichia coli
FEBS Journal, 2000Co-Authors: Ashok K Patra, Gagandeep Kaur Gahlay, S K Gupta, Vijaya B V Reddy, Amulya K PandaAbstract:An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca Radiata) zona pellucida glycoprotein-C (bmZPC), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned in pQE-30 vector and the protein expressed as inclusion bodies in Escherichia coli. Recombinant bmZPC (r-bmZPC) was solubilized from purified inclusion bodies in the absence of a high concentration of chaotropic agents and was subsequently refolded. Use of a low concentration of urea (2 m) during solubilization of r-bmZPC helped to minimize the extent of protein aggregation during refolding of the recombinant protein, and retain the existing native-like secondary structure that was essential for proper folding. Purified r-bmZPC appeared as a dominant band of 43 kDa on SDS/PAGE and Western blot. Although it lacked carbohydrate moieties, the purified and refolded r-bmZPC bound to the head region of bonnet monkey spermatozoa, confirming the existence of a native-like conformation. CD revealed a maximum at 200 nm and a single broad minimum extending from 209 to 216 nm, indicating the presence of both α-helical and β-sheet conformations in the refolded r-bmZPC. Two different phases of transition were observed by urea-gradient electrophoresis, suggesting the existence of multiple intermediate stages during the unfolding of r-bmZPC. The availability of refolded r-bmZPC will help in elucidating its role during the complex cascade of events during fertilization.
Renuka Kaul - One of the best experts on this subject based on the ideXlab platform.
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immunocontraceptive potential of recombinant bonnet monkey Macaca Radiata zona pellucida glycoprotein c expressed in escherichia coli and its corresponding synthetic peptide
Reproductive Biomedicine Online, 2001Co-Authors: Renuka Kaul, Chhabi K Govind, Neela Sivapurapu, Abhijit Afzalpurkar, V Srikanth, S K GuptaAbstract:Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for development of immunocontraceptive vaccines. In this study, the efficacy to block fertility by immunization with recombinant bonnet monkey (Macaca Radiata) zona pellucida glycoprotein-C (r-bmZPC) expressed in Escherichia coli and its synthetic peptide (P4: KGDCGTPSHSRRQPHVVSQWSRSA, aa residues 324-347) conjugated to diphtheria toxoid (DT) has been evaluated in a homologous system. Female bonnet monkeys, immunized with P4-DT conjugate showed better immunocontraceptive potential as compared to an r-bmZPC-DT immunized group. In spite of high anti-P4 antibody titres, animals continued to have ovulatory cycles and showed no disturbance in cyclicity (except summer amenorrhoea). No ovarian pathology was observed in the P4 immunized group. These results suggest that immunization with the P4 may lead to block in fertility without obvious ovarian dysfunction. However, further inputs are required to identify additional ZP based B-cell epitopes to enhance the contraceptive efficacy.
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Sequence of complementary deoxyribonucleic acid encoding bonnet monkey (Macaca Radiata) zona pellucida glycoprotein-ZP1 and its high-level expression in Escherichia coli.
Biology of Reproduction, 1997Co-Authors: Satish K Gupta, Manju Sharma, Aruna K. Behera, Rachna Bisht, Renuka KaulAbstract:Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for immunocontraception. Studies on this potential use can be facilitated by the availability of recombinant proteins. A cDNA lambda gt11 library was constructed using poly(A)+ mRNA isolated from bonnet monkey (Macaca Radiata) ovaries and was screened for bonnet monkey ZP1 using a 404-basepair (bp) human ZP1 fragment (nucleotides 818-1221) as probe. Bonnet monkey ZP1 cDNA comprises 1617 nucleotides and encodes a polypeptide of 539 amino acid residues that share 92.0% identity with human ZP1. The major difference between bonnet monkey ZP1 and human ZP1 is the deletion of a 28-amino acid domain (amino acid residues 100-127 corresponding to human ZP1). An internal fragment (1317 bp) of bonnet monkey ZP1, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by polymerase chain reaction. The amplified Sac I and Kpn I restricted fragment was cloned in a frame downstream of the T5 promoter under the lac operator control for expression in the pQE-30 vector. Recombinant ZP1 (r-ZP1) was expressed as a polyhistidine fusion protein in Escherichia coli strains SG13009[pREP4] and ompT and Ion protease-deficient BL21 (plysS). SDS-PAGE analysis and immunoblotting with a murine monoclonal antibody, MA-410 (raised against porcine ZP3alpha--a homologue of bonnet monkey ZP1--and cross-reactive with bonnet monkey zona pellucida), revealed major bands of 51 and 40 kDa besides truncated fragments. Optimum expression of r-ZP1 was observed at 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG). Immunization of male rabbits with r-ZP1 purified on nickel-nitrilotriacetic acid (NTA) resin under denaturing conditions and of female rabbits with r-ZP1 conjugated with diphtheria toxoid-generated antibodies reactive with r-ZP1 in ELISA. Moreover, immune sera, when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The information on the sequence of bonnet monkey ZP1 and the availability of the recombinant protein will help toward better understanding and evaluation of the contraceptive potential of homologous immunization in a nonhuman primate model.
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expression of bonnet monkey Macaca Radiata zona pellucida 3 zp3 in a prokaryotic system and its immunogenicity
Molecular Reproduction and Development, 1997Co-Authors: Renuka Kaul, Abhijit Afzalpurkar, S K GuptaAbstract: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 SacI restricted fragment was cloned in frame 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 ZP3 beta-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 ZP3 beta and bonnet r-ZP3 but failed to react with porcine ZP3 alpha 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.
Chhabi K Govind - One of the best experts on this subject based on the ideXlab platform.
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Purified and Refolded Recombinant Bonnet Monkey (Macaca Radiata) Zona Pellucida Glycoprotein-B Expressed in Escherichia coli Binds to Spermatozoa1
2016Co-Authors: Chhabi K Govind, Sangeeta Choudhury, Eep K. Gahlay, Satish K GuptaAbstract:Bonnet monkey (Macaca Radiata) zona pellucida glycopro-tein-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 re-folded r-bmZPB without urea and devoid of lower molecular weight fragments was achieved by following an alternate meth-odology that involved purification of inclusion bodies to ho-mogeneity and solubilization in the presence of a low concen-tration 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 re-vealed the presence of both a helical and b 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 an-tibodies generated against r-bmZPB. These results suggest that nonglycosylated bmZPB binds to capacitated as well as acro-some-reacted spermatozoa in a nonhuman primate and may have a functional role during fertilization. fertilization, ovum, sper
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purified and refolded recombinant bonnet monkey Macaca Radiata zona pellucida glycoprotein b expressed in escherichia coli binds to spermatozoa
Biology of Reproduction, 2001Co-Authors: Chhabi K Govind, Gagandeep Kaur Gahlay, Sangeeta Choudhury, S K GuptaAbstract:Abstract 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 α 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 assa...
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immunocontraceptive potential of recombinant bonnet monkey Macaca Radiata zona pellucida glycoprotein c expressed in escherichia coli and its corresponding synthetic peptide
Reproductive Biomedicine Online, 2001Co-Authors: Renuka Kaul, Chhabi K Govind, Neela Sivapurapu, Abhijit Afzalpurkar, V Srikanth, S K GuptaAbstract:Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for development of immunocontraceptive vaccines. In this study, the efficacy to block fertility by immunization with recombinant bonnet monkey (Macaca Radiata) zona pellucida glycoprotein-C (r-bmZPC) expressed in Escherichia coli and its synthetic peptide (P4: KGDCGTPSHSRRQPHVVSQWSRSA, aa residues 324-347) conjugated to diphtheria toxoid (DT) has been evaluated in a homologous system. Female bonnet monkeys, immunized with P4-DT conjugate showed better immunocontraceptive potential as compared to an r-bmZPC-DT immunized group. In spite of high anti-P4 antibody titres, animals continued to have ovulatory cycles and showed no disturbance in cyclicity (except summer amenorrhoea). No ovarian pathology was observed in the P4 immunized group. These results suggest that immunization with the P4 may lead to block in fertility without obvious ovarian dysfunction. However, further inputs are required to identify additional ZP based B-cell epitopes to enhance the contraceptive efficacy.
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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
Biology of Reproduction, 2000Co-Authors: Chhabi K Govind, Akiko Hasegawa, Koji Koyama, S K GuptaAbstract: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.
Satish K Gupta - One of the best experts on this subject based on the ideXlab platform.
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Purified and Refolded Recombinant Bonnet Monkey (Macaca Radiata) Zona Pellucida Glycoprotein-B Expressed in Escherichia coli Binds to Spermatozoa1
2016Co-Authors: Chhabi K Govind, Sangeeta Choudhury, Eep K. Gahlay, Satish K GuptaAbstract:Bonnet monkey (Macaca Radiata) zona pellucida glycopro-tein-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 re-folded r-bmZPB without urea and devoid of lower molecular weight fragments was achieved by following an alternate meth-odology that involved purification of inclusion bodies to ho-mogeneity and solubilization in the presence of a low concen-tration 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 re-vealed the presence of both a helical and b 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 an-tibodies generated against r-bmZPB. These results suggest that nonglycosylated bmZPB binds to capacitated as well as acro-some-reacted spermatozoa in a nonhuman primate and may have a functional role during fertilization. fertilization, ovum, sper
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Bonnet monkey (Macaca Radiata) ovaries, like human oocytes, express four zona pellucida glycoproteins.
Molecular Reproduction and Development, 2007Co-Authors: Anasua Ganguly, R.k. Sharma, Satish K GuptaAbstract:Recent studies document that the zona pellucida matrix of human oocytes is composed of four glycoproteins designated as ZP1, ZP2, ZP3, and ZP4 instead of three as proposed in mouse model. In the present study, investigations were carried out to find the presence of the fourth ZP glycoprotein in the ovaries of non-human primates namely bonnet monkey (Macaca Radiata). Employing total RNA isolated from bonnet monkey ovaries, the complementary deoxyribonucleic acid (cDNA) encoding bonnet monkey ZP1 (up to furin cleavage site) was successfully amplified by reverse transcribed polymerase chain reaction (RT-PCR). The deduced amino acid (aa) sequence of bonnet monkey ZP1 revealed 96.0% identity with human ZP1. The 21 cysteine residues present in bonnet monkey ZP1 were conserved in human, mouse, rat, quail, and chicken. Simultaneously, polyclonal antibodies were generated in mice against synthetic peptides corresponding to human ZP1 (P1, 137–150 aa; P2, 223–235 aa; P3, 237–251 aa; P4, 413–432 aa; and P5, 433–451 aa). Employing anti-peptide antibodies that were devoid of cross-reactivity as determined by ELISA with human/bonnet monkey recombinant ZP2, ZP3, and ZP4, the presence of ZP1 in the ovaries of bonnet monkey and human oocytes was demonstrated by indirect immunofluorescence. The antibodies against peptides P3 and P4 reacted only with the ZP of bonnet monkey ovaries and not other ovarian associated cells. The data presented in this manuscript provide evidence, for the first time, that the bonnet monkey ZP matrix is composed of four glycoproteins. Mol. Reprod. Dev. 75: 156–166, 2008. © 2007 Wiley-Liss, Inc.
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Sequence of complementary deoxyribonucleic acid encoding bonnet monkey (Macaca Radiata) zona pellucida glycoprotein-ZP1 and its high-level expression in Escherichia coli.
Biology of Reproduction, 1997Co-Authors: Satish K Gupta, Manju Sharma, Aruna K. Behera, Rachna Bisht, Renuka KaulAbstract:Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for immunocontraception. Studies on this potential use can be facilitated by the availability of recombinant proteins. A cDNA lambda gt11 library was constructed using poly(A)+ mRNA isolated from bonnet monkey (Macaca Radiata) ovaries and was screened for bonnet monkey ZP1 using a 404-basepair (bp) human ZP1 fragment (nucleotides 818-1221) as probe. Bonnet monkey ZP1 cDNA comprises 1617 nucleotides and encodes a polypeptide of 539 amino acid residues that share 92.0% identity with human ZP1. The major difference between bonnet monkey ZP1 and human ZP1 is the deletion of a 28-amino acid domain (amino acid residues 100-127 corresponding to human ZP1). An internal fragment (1317 bp) of bonnet monkey ZP1, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by polymerase chain reaction. The amplified Sac I and Kpn I restricted fragment was cloned in a frame downstream of the T5 promoter under the lac operator control for expression in the pQE-30 vector. Recombinant ZP1 (r-ZP1) was expressed as a polyhistidine fusion protein in Escherichia coli strains SG13009[pREP4] and ompT and Ion protease-deficient BL21 (plysS). SDS-PAGE analysis and immunoblotting with a murine monoclonal antibody, MA-410 (raised against porcine ZP3alpha--a homologue of bonnet monkey ZP1--and cross-reactive with bonnet monkey zona pellucida), revealed major bands of 51 and 40 kDa besides truncated fragments. Optimum expression of r-ZP1 was observed at 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG). Immunization of male rabbits with r-ZP1 purified on nickel-nitrilotriacetic acid (NTA) resin under denaturing conditions and of female rabbits with r-ZP1 conjugated with diphtheria toxoid-generated antibodies reactive with r-ZP1 in ELISA. Moreover, immune sera, when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The information on the sequence of bonnet monkey ZP1 and the availability of the recombinant protein will help toward better understanding and evaluation of the contraceptive potential of homologous immunization in a nonhuman primate model.
