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Molecular characterization of zona pellucida glycoproteins: role in fertilization and regulation of fertility |
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Principal Investigator : Satish K Gupta Project Associates/
Assistants PhD Students Collaborators Major themes of this
project are A) Molecular and biochemical characterization of zona pellucida (ZP)
glycoproteins and delineation of their role during fertilization, and B)
Evaluation of the immunocontraceptive potential of ZP based immunogens. The
objectives of the study are (i) expression of non-human primate and canine ZP
glycoproteins in various expression systems, (ii) binding characteristics of
purified recombinant ZP proteins to spermatozoa in a non-human primate model,
(iii) design non-human primate ZP glycoproteins based synthetic peptides and
evaluate their immunogenicity, (iv) evaluate immunocontraceptive potential of
non-human primate and canine recombinant ZP proteins for fertility regulation
in respective homologous animal models and (v) characterization of the immune
response to plasmid DNA encoding ZP glycoproteins and rabies glycoprotein-G. Cloning
and expression of non-human primate and canine zona pellucida proteins in
baculovirus expression system To
express the glycosylated form of r-bmZP2, the full length cDNA (1.8 kb),
excluding the N-terminal signal sequence and C-terminal transmembrane-like
domain was PCR amplified and cloned downstream of a late polyhedrin promoter
in BioBlueTM-His6 baculovirus transfer vector under EcoR I-Bgl
II restriction sites. The plasmid DNA (BioBlueTM-bmZP2) was co-transfected with BaculoGoldTM baculovirus DNA in Sf9 cells and a positive
plaque was picked up for further studies. The recombinant protein was
expressed as a fusion protein with blue fluorescence protein (BFP) and His6 tag. Time course experiments showed that the
maximum expression of the protein occurred 72 hr post infection. The
recombinant protein was purified by Ni-NTA column in the presence of urea,
which was dialyzed out in renaturing buffer. It showed an apparent molecular
weight of 100 kDa in western blot. Similarly,
recombinant dog ZP2 (r-dZP2) and dog ZP3 (r-dZP3) glycoproteins were also
expressed as BFP-His6
fusion proteins in Sf9 cells. The expressed proteins showed a band
corresponding to 100 and 66 kDa respectively in western blot. Attempts are
being made to purify r-dZP2/r-dZP3 and to optimize conditions for cleavage of
the BFP-His6
tag from the expressed r-dZP2/r-dZP3. Structural
elucidation of r-bmZP3 As
reported last year, we were able to purify the E. coli expressed
r-bmZP3 in the absence of chaotropic agents. Further to that, it was subjected
to the following circular dichroism (CD) spectroscopic studies to evaluate its
secondary structural attributes. All the CD spectra were recorded using a 0.1
cm path length cuvette in 20 mM Tris buffer, pH 8.5 in a spectral range from
195-260 nm. The r-bmZP3 showed a shift in the spectral minima, when the
concentration was lowered from 25 mM to 12.5 mM.
At 50 and 25 mM concentrations, the protein showed a
minima around 225 nm, which shifted to 212 nm at 12.5 mM
concentration and remained unchanged till 3 mM. At 12.5 mM
concentration, the protein showed a predominantly b
sheet structure, though a reasonable a helical component
was also observed. In
order to further investigate the structure-activity relationship of bmZP3
protein, computer aided similarity searches across various species were
performed. As a result, a core fragment of the Cloning
and expression of recombinant rabies virus glycoprotein-G (r-rG) in E. coli
and baculovirus expression system In
order to clone rabies virus glycoprotein G (rG), BHK21 cells were infected
with PM10 strain of rabies virus. Total RNA from the infected cells was
prepared at various time periods post – infection using TRIZOL reagent.
Total RNA was directly used to amplify the cDNA corresponding to r-rG without
signal sequence and transmembrane like domain by RT-PCR which resulted in the
amplification of a 1.313 kb fragment. Comparison of the nucleotide sequence of
the cloned rG with sequences from Gene Bank revealed an identity with other
strains ranging from 86-99.9%. The deduced amino acid sequence revealed 100%
identity with the sequence having an Accession No. M81058. The 1.313 kb
fragment was excised with Bam H I and Kpn I restriction
enzymes from PCR-Script SK (+)-rG clone, purified and cloned in-frame
downstream of His6 tag under T5 promoter – lac operator control
in pQE30 expression vector. Positive clones were identified and checked for
expression of r-rG by SDS-PAGE and immunoblotting using a commercially
available human rabies immunoglobulin. The western blot analysis revealed a
band of approximately 55 kDa. The r-rG protein was purified under denaturing
conditions by Ni-NTA affinity chromatography and antibodies were generated
against the recombinant protein in male New Zealand white rabbit. In
order to obtain r-rG in glycosylated form, it was expressed in baculovirus
expression system. The pQE-rG plasmid was used as a template to PCR amplify
cDNA corresponding to rG with a forward primer incorporating BamH I
site and the reverse primer incorporating EcoR I site. The BamH I-EcoR
I restricted fragment was cloned into pAcSecG2T vector under the late
polyhedrin promoter. The r-rG was expressed as a GST fusion protein wherein
the native signal sequence was replaced with the insect signal sequence (gp67)
in Sf9 cells. The r-rG showed a band with an apparent molecular weight of 75
kDa in western blot. Attempts are underway to purify r-rG using the
glutathione agarose beads and to optimize the conditions for thrombin
cleavage. Immunogenicity
of ZP glycoproteins based synthetic peptides The
synthetic peptide immunogens capable of generating antibodies that reacted
with native ZP and inhibited sperm-egg interaction corresponding to bmZP1 were
reported last year. In addition, 5 synthetic peptides corresponding to bmZP2
(bmZP2(88-102 aa), bmZP2(103-125 aa), bmZP2(124-143 aa), bmZP2(395-407 aa) and bmZP2(532-549 aa)) were made and conjugated
individually to DT at a 10:1 molar ratio. Further, two synthetic peptides
corresponding to human (h) ZP2 (hZP2(85-109
aa); has 5 changes in aa as compared to bmZP2 sequence and hZP2(103-125 aa); having 3 changes in aa
as compared to bmZP2 sequence) were also conjugated to DT. Female BALB/cJ mice
were immunized with the individual peptide-DT conjugates. The antisera against
bmZP2(124-143 aa), bmZP2(395-407 aa) and bmZP2(532-549 aa) have been analyzed for
their recognition of the respective peptide and E. coli expressed
r-bmZP2 in an ELISA. Anti-peptide antibodies were generated against the
respective peptides but only anti-bmZP2(532-549
aa) recognized r-bmZP2 in an ELISA and also recognized native bonnet
monkey zona. The antibodies against hZP2(103-125
aa) evaluated so far, recognized human ZP and also inhibited sperm-egg
interaction. A
chimeric protein bmZP13 encompassing bmZP1(246-278
aa) (comprising the aa stretch 248-273, antisera against which inhibited
human sperm-egg binding) and bmZP3(324-347
aa) (immunization with this peptide in a homologous model showed block
in fertility without concomitant ovarian pathology) was made at the cDNA
level, with a tri-glycine spacer separating the individual epitopes. The
bmZP13 cDNA was cloned in frame of the polyhistidine tag, downstream of the T5
promoter in pQE30 expression vector. The purification of r-bmZP13 is in
progress. Previously,
we have reported expression of the chimeric recombinant protein (r-bmZP123)
encompassing the B cell epitopes of bmZP1(132-147
aa), bmZP2(86-113 aa)
and bmZP3 (324-347 aa) in E. coli. Analysis of the rabbit polyclonal antibodies
generated against r-bmZP123 revealed variable reactivity with the native ZP
and also in vitro contraceptive efficacy. The r-bmZP123 has 4 cysteine
residues, which may lead to different inter and intra-molecular disulphide
bond formations that may influence the antibody response. In order to
circumvent this, r-bmZP123 was reduced and the cysteine residues were
chemically blocked with iodoacetamide. The reduced and iodoacetamidated
r-bmZP123 was conjugated to DT using the ‘two step’ glutaraldehyde method.
Two female rabbits (R-67, R-68) were immunized with r-bmZP123-DT conjugate.
Antibodies were generated against r-bmZP123, r-bmZP1, r-bmZP2 and r-bmZP3 as
determined by ELISA. The sera from both the female rabbits showed reactivity
with the whole native bonnet monkey ZP. Characterization of the
immune response to plasmid DNA encoding ZP glycoproteins and rabies
glycoprotein-G Last
year, we had reported cloning of bmZP1 cDNA in VR1020 mammalian expression
vector (VRbmZP1). Expression of bmZP1 in COS-1 cells was confirmed by flow
cytometric analysis and western blot analysis which revealed a protein band of
approximately 57 kDa as compared to the calculated molecular weight of 51.4
kDa (including TPA signal sequence), suggesting that ZP1 may be expressed in a
glycosylated form. The immunogenicity of VRbmZP1 was evaluated by immunizing
male BALB/cJ mice intramuscularly (i.m.) with 100 µg DNA/mouse in saline
administered thrice on days 0, 21 and 35. Mice immunized with VRbmZP1 plasmid
DNA showed significantly higher anti-r-bmZP1 antibodies in comparison to
vector immunized mice as determined in an ELISA employing r-bmZP1 expressed in
E. coli. The anti-bmZP1 antibodies generated were specific to zona as
evident by positive immunofluorescence of VRbmZP1 immunized mice sera with
bonnet monkey ovarian sections in an indirect immunofluorescence assay. Also
these antibodies inhibited the binding of human spermatozoa to the antibody
treated human zona in a hemizona assay thus indicating their
immunocontraceptive potential. After
achieving some success with the bmZP1 model, the cDNA corresponding to dZP3,
excluding the N-terminal signal sequence and C-terminus transmembrane-like
domain, was also cloned in VR1020 (VRdZP3). The immunogenicity of VRdZP3 was
analyzed by three different modes of plasmid DNA delivery in male BALB/cJ
mice- a) intramuscular (i.m.) administration of DNA in saline, b) i.m.
administration of DNA in saline followed by electroporation wherein short
electric pulses were given at the site of injection, and c) intradermal
administration of DNA entrapped in gold microcarriers and delivered by gene
gun. A good antibody response was observed by all the three modes of
immunization as determined in an ELISA employing r-dZP3 expressed in E. coli.
The anti-dZP3 antibodies also recognized native ZP in an indirect
immunofluorescence assay. Interestingly, female BALB/cJ mice immunized with
VRdZP3 plasmid DNA by gene gun method, generated antibody response similar to
the male mice. These are interesting observations of generating antibodies
against ZP3 without coupling to a carrier. The status of fertility of VRdZP3
immunized female mice is under observation. In
the last year report, it was also proposed to make a hybrid construct of dZP3
with rabies glycoprotein-G (rG) with the aim of generating immune response
against ZP3 that may block fertility and rG that may provide protection
against rabies. Before initiating the making of plasmid DNA construct encoding
dZP3-rG hybrid construct, it was pertinent to study immune response to a
plasmid DNA encoding rG alone. Two constructs were made using VR1020 vector- (i)
rG cDNA excluding the native signal sequence and the transmembrane- like
domain (rGVR), (ii) rG cDNA excluding the native signal sequence and retaining
the transmembrane- like domain (rGVRt). Localization studies revealed that the
expression of rG with both the constructs was cytosolic. The presence of the
transmembrane domain in rG, expressed by rGVRt plasmid DNA, did not lead to
anchoring of the protein on the cell membrane. However, rGVRt plasmid DNA
elicited higher antibody response as compared to rGVR plasmid DNA, thereby
suggesting that the transmembrane- like domain may enhance humoral response
against r-rG. The influence of native signal sequence on protein trafficking
is being evaluated. Publications Original
peer-reviewed articles 1.
Srivastava N, Santhanam R, Sheela P, Mukund S, Thakral SS, Malik BS and
Gupta SK (2002) Evaluation of the immunocontraceptive potential of Escherichia
coli expressed recombinant dog ZP2 and ZP3 in homologous animal model. Reproduction
(in press). 2.
Rath A, Choudhury S, Hasegawa A, Koyama K and Gupta SK (2002)
Antibodies generated in response to plsmid DNA encoding zona pellucida
glycoprotein-B inhibit in vitro human sperm-egg binding. Mol Rep Dev
(in press). 3.
Koyama K, Hasegawa A and Gupta SK (2002) Prospect for
immunocontraception using the NH2-terminal recombinant peptide of human zona
pellucida protein-A (hZPA). Am J Reprod Immunol (in press). 4.
*Gahlay GK, Srivastava N, Govind CK and Gupta SK (2002) Primate
recombinant zona pellucida proteins expressed in Escherichia coli binds
to spermatozoa. J Reprod Immunol 53:67-77 (*in press last year,
since published). 5.
*Kaul R, Sivapurapu N, Afzalpurkar A, Srikant V, Govind CK and Gupta SK
(2001) Immunocontraceptive potential of recombinant bonnet monkey (Macaca
radiata) zona pellucida glycoprotein-C expressed in Escherichia coli
and its corresponding synthetic peptide. Reproductive BioMedicine Online
2:33-39 (*in press last year, since published). 6.
Sivapurapu N, Upadhyay A, Hasegawa A, Koyama K and Gupta SK (2002)
Native zona pellucida reactivity and in vitro effect on human sperm-egg
binding with antisera against bonnet monkey ZP1 and ZP3 synthetic peptides. J
Reprod Immunol (in press). Reviews/Proceedings 1.
Upadhyay A, Sivapurapu N, Rath A and Gupta SK (2001) Rationale of
synthetic peptides as immunogens for vaccine development. Proc Indian Natl
Sci Acad B (in press). Patents 1. Gupta SK, Kalsi G, Shamim M, Nataraju B and Datta RK. A process for
preparing diagnostic monoclonal antibody based system for the rapid detection
of nuclear polyhedrosis in Bombyx mori l. Indian patent No. 186538
granted in Sep 2001. 2. Gupta SK, Shamim M, Ghosh D, Baig M, Nataraju B and Datta RK. A process
for producing monoclonal antibodies coated latex beads for the detection of
pebrine infection in Bombyx mori l. Indian patent No. 186722 granted
in Oct 2001. 3. Gupta SK, Sharma M, Behera AK, Bisht R and Kaul R. A method of inducing
infertility in a mammal, a recombinant protein for inducing such infertility
and a process for producing the recombinant protein. Indian patent No.
186770 granted in Nov 2001. |