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Molecular characterization of
zona pellucida glycoproteins: role in fertilization and regulation of fertility |
| Principal Investigator : Satish K Gupta
Project Associates
/ Assistants Ph D Students Collaborators The major themes of this group are molecular and biochemical characterization of zona pellucida (ZP) glycoproteins, delineation of their role during fertilization, and evaluation of the immunocontraceptive potential of ZP-based immunogens. The objectives of the study are (i) expression of human, bonnet monkey and canine ZP glycoproteins in various expression systems, (ii) delineation of structural and functional attributes of purified recombinant ZP proteins/glycoproteins, and (iii) immunization strategies aiming to inhibit fertility and provide protection against rabies infection. I
Expression of ZP glycoproteins in baculovirus expression system and their
purification This
year, our group has initiated the expression of human ZP glycoproteins, both
in E. coli as well as baculovirus expression systems. The cDNA
corresponding to human ZP2 (hZP2) and ZP3 (hZP3) were a kind gift from Dr.
Jurrien Dean, Laboratory of Cellular and Developmental Biology, National
Institutes of Health, USA. The cDNA corresponding to human ZP1 (hZP1) was made
available by Dr. Sarvamangala V. Prasad, Department of Molecular and Cellular
Biology, Baylor College of Medicine, USA. Initially r-hZP1 (23-490 aa), r-hZP2
(38-590 aa) and r-hZP3 (23-348 aa) were expressed in E.coli as His6-tag
fusion protein using pRSET-A expression vector. To express the glycosylated
form of r-hZP3, the full length cDNA (1272 bp; 1-424 aa), including the
N-terminal signal sequence and the C-terminal transmembrane-like domain was
PCR amplified and cloned downstream of the late polyhedrin promoter in pAcHLT-A
transfer vector. The plasmid DNA (pAcHLTA-hZP3) was co-transfected along with
baculovirus DNA (BaculoGoldTM)
into Sf21 cells and positive plaques were picked up for further
studies. The recombinant protein was expressed as a fusion protein with a His6-tag
at the N-terminus. The r-hZP3 was purified by using Ni-NTA resin in the
presence of 8M urea, which was renatured in a buffer without any chaotropic
agent by dialysis. The purified r-hZP3 showed an apparent molecular weight of
approximately 60 kDa in Western blot. Similarly, r-hZP1 (1-566 aa) and r-hZP2 (1-746 aa) have also been expressed as His6-fusion proteins in Sf21 cells. The purified r-hZP1 and r-hZP2 showed a molecular weight of ~80 kDa and ~110 kDa respectively in Western blot. Non-human primate ZP glycoproteins Last
year, we had reported the cloning and expression of r-bmZP2 in the baculovirus
expression system as a fusion protein with blue fluorescent protein (BFP) and
His6-tag
at the N-terminus. In the present year of report, the full length cDNA of
bmZP3 (978 bp) and bmZP1 (1317 bp), excluding the N-terminus signal sequence
and C-terminus transmembrane like-domain, have also been similarly cloned and
expressed in the baculovirus expression system under the control of late
polyhedrin promoter using the BioBlueTM-His6
baculovirus transfer vector. The expressed recombinant proteins, after
purification using Ni-NTA column chromatography and subsequent dialysis in
refolding buffer, showed a band corresponding to ~66 kDa for bmZP3 and ~80 kDa
for bmZP1. In addition, bmZP3 has also been cloned in another baculovirus
transfer vector, pAcHLT-A, as a His6-fusion
protein, devoid of the BFP. The purified r-bmZP3 without BFP showed an
apparent molecular weight of ~ 52 kDa. Characterization
of the nature of glycosylation by using lectin binding assay in ELISA revealed
that all the three recombinant proteins have chiefly N-linked type of
glycosylation. The r-bmZP2 showed binding with PNA suggesting galactose
residue as main source of glycosylation whereas r-bmZP1 and r-bmZP3 bound
strongly to both ConA and PNA suggesting the presence of mannose and galactose. We
have reported earlier the expression of r-dZP2 and r-dZP3 as BFP-His6-fusion
proteins in baculovirus expression system. During this year, we have tried to
optimize the conditions for purification of the r-dZP3 protein and cleavage of
BFP-His6-tag
from the purified r-dZP3. In this direction, the Ni-NTA purified fusion
protein was treated with thrombin under different experimental conditions with
respect to temperature, time, inclusion of heparin and CaCl2
in
the cleavage buffer, to cleave the BFP-His6-tag
at the thrombin cleavage site. None of the above experimental conditions
yielded an appreciable amount of r-dZP3 without the BFP-His6-tag. In
light of the above results, the cDNA corresponding to dZP2 and dZP3 were
further cloned in pAcHLT-A baculovirus transfer vector, which unlike the
BioBlueTM
has only a His6-tag
at the N-terminus. The expression of r-dZP3 and r-dZP2 fusion proteins in Sf21
cells were checked by Western blot which revealed the presence of a ~45 kDa
band corresponding to r-dZP3 and ~80 kDa band corresponding to r-dZP2 in the
cell lysate. The r-dZP3 glycoprotein was purified using Ni-NTA resin and
obtained in the absence of any chaotropic agent. Binding characteristics of recombinant ZP glycoproteins to spermatozoa Availability of the purified r-bmZP1, r-bmZP2 and r-bmZP3, expressed in baculovirus, allowed us to analyze their binding characteristics with the capacitated and acrosome-reacted bonnet monkey spermatozoa. The purified r-bmZP1 bound to the head region of capacitated spermatozoa and showed binding to the equator, mid piece and tip of the head of the acrosome-reacted spermatozoa. The r-bmZP2 also showed the same binding pattern as r-bmZP1 with both capacitated and acrosome-reacted spermatozoa. The r-bmZP3 bound to the principal segment of the head of the capacitated spermatozoa and failed to show any binding to the acrosome-reacted spermatozoa. Glycosylation of ZP3 – the primary sperm receptor is critical for induction of acrosomal exocytosis Recombinant
bmZP3 expressed in E. coli as well as in baculovirus expression system
were also examined for their ability to induce acrosome reaction in bonnet
monkey spermatozoa. The recombinant protein (25 nmoles) was incubated with 1 X
106
capacitated sperm in a total reaction volume of 200 ml
for 18 h and the sperm were analyzed for its acrosomal status by staining with
tetramethylrhodamine isothiocyanate (TRITC) conjugated Pisum sativum
agglutinin (PSA). Only the glycosylated r-bmZP3 was able to induce acrosome
reaction, which was significantly higher as compared to the control (~30%).
The E. coli expressed r-bmZP3 failed to induce any significant level of
acrosomal exocytosis. Similarly, baculovirus expressed r-hZP3 also induced acrosomal exocytosis in the capacitated human spermatozoa, whereas the E. coli expressed protein failed to do so. The induction of acrosomal exocytosis, in this case was observed as early as 30 min after incubation with r-hZP3. As low as 100 ng/ml of r-hZP3 induced significant increase in acrosomal exocytosis as compared to control. Identification
and characterization of the ZP3 fragment capable of binding to the capacitated
spermatozoa III In vitro contraceptive efficacy of polyclonal antibodies generated against chimeric recombinant protein and synthetic peptides based on ZP glycoproteins During
the year of report, in collaboration with Prof. Koji Koyama, Hyogo College of
Medicine, Japan, we have demonstrated that rabbit polyclonal antibodies
against chimeric r-bmZP123 not only reacted with the native human ZP but also
inhibited, in vitro, the binding of human spermatozoa to ZP in a
hemizona assay. We have also synthesized a chimeric peptide encompassing
bmZP1(251-273
aa)
(comprising the aa stretch 251-273, 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) epitopes, with a tri-glycine IV Characterization of the immune response to plasmid DNA encoding dZP3 and rabies glycoprotein-G To
optimize the DNA vaccine encoding rabies glycoprotein-G (rG), the following 4
constructs were made using VR1020 vector: (i) rG cDNA excluding the native
signal sequence (SS) and the transmembrane-like domain (TD; rGVR), (ii) rG
cDNA excluding the native SS and retaining the TD (rGVRt), (iii) rG cDNA
including the native SS and excluding the TD (rGVRs), and (iv) rG cDNA
including both native SS and TD (rGVRst). In vitro transfection
of COS-1 mammalian cells with each of rG plasmid DNA construct revealed
expression of rG as analyzed by indirect immunofluorescence, flow cytometry
and Western blot. Immunogenicity studies in mice revealed that rGVRt plasmid
DNA elicited highest antibody response. Mice immunized with rGVRt plasmid DNA
also showed the highest rabies virus neutralizing antibody (RVNA) response as
compared to the other three constructs as analyzed by an in vitro Rapid
Fluorescent Focus Inhibition Test (RFFIT). In addition to humoral immune
response, all the four constructs also elicited cell mediated immune response.
Subsequently, DNA vaccine construct employing VR1020 vector and encoding
dZP3-rG fusion protein has also been made (VRdZP3-rG). Transfection of COS-1
cells, in vitro, with VRdZP3-rG plasmid DNA construct revealed the
cytosolic presence of both dZP3 as well as rG. Western blot studies showed a
band corresponding to ~90 kDa. Though the VRdZP3-rG plasmid DNA is immunogenic,
but none of the immunized mice elicited protective RVNA response. Expression of rabies glycoprotein–G in baculovirus expression system Previously,
we have reported the expression in baculovirus of recombinant rG (r-rG) as a
GST fusion protein using pAcSecG2T transfer vector. In the current year,
attempts to purify recombinant GST-rG fusion protein and subsequent cleavage
of GST did not yield the desirable amount of r-rG. In view of this, an alternate expression vector (pAcHLT-A) was employed. The cDNA corresponding to rG including signal sequence and transmembrane-like domain was PCR amplified from pKB3-JE-13 clone (ATCC, Rockville, MD, USA) which encoded the full length rG of Challenge Virus Standard (CVS) strain of rabies. The amplified product was cloned into the pGEM-T cloning vector and subsequently cloned into pAcHLT-A vector under the late polyhedrin promoter. Expression of r-rG glycoprotein in Sf21 cells was checked by Western blot analysis, which revealed the presence of ~60 kDa band in the cell lysate. The recombinant protein was purified by Ni-NTA column in the presence of urea followed by renaturation in a buffer without urea. Expression of fusion protein corresponding to dZP3-rG and dZP2-rG in baculovirus expression system To
construct the hybrid cDNA encoding dZP3 (978 bp) and rG (1440 bp), these were
PCR amplified individually using appropriate primers from the respective
parent clones (pQE-30-dZP3 and pKB3-JE-13). The PCR amplified and purified
products were used to assemble the hybrid cDNA encoding dZP3-rG (2360 bp) by a
second PCR. The assembled PCR product was cloned into pGEM-T vector followed
by cloning in pAcHLT-A baculovirus transfer vector. The pAcHLTA-dZP3-rG
construct was co-transfected along with BaculogoldTM
DNA as described before and the recombinant protein was expressed in Sf21
cells. The purified r-dZP3-rG fusion protein revealed a band of ~100 kDa in
Western blot. To make the hybrid cDNA construct encoding dZP2 and rG, the cDNAs encoding dZP2 (1800 bp) and rG (1440 bp) were PCR amplified separately using appropriate primers from the respective parent clones (pQE-30-dZPA and pKB3-JE-13). Each of the PCR amplified and purified product was cloned into pCR-Script SK(+) cloning vector. The positive pCR-Script-dZP2 clone restricted with Sac I and Not I site was further cloned into pAcHLT-A baculovirus transfer vector. Not I and Kpn I restricted fragment of positive pCR-Script-rG clone was then subsequently cloned into pAcHLT-A-dZP2 plasmid. Expression of r-dZP2-rG was carried out in a similar fashion as r-dZP3-rG fusion protein which revealed the presence of ~130 kDa band by Western blot analysis. The purification of r-dZP2-rG protein is under way. Immunization of female dogs The
active immunization studies of non-descript female dogs have been initiated
this year to evaluate the feasibility of developing immunization strategy for
blocking fertility and to simultaneously generate rabies virus neutralization
antibodies. The female dogs (n = 13) used in the present study have been
reared at Central Military Veterinary Laboratory, Meerut Cantt. Animals were
divided randomly into three groups. The female dogs were immunized
intramuscularly at two sites with r-dZP3-rG (n=5; equivalent to 250 mg
r-dZP3-rG per animal), physical mixture of r-dZP3-DT and r-rG (n=5; equivalent
to 250 mg
r-dZP3-DT and 250 mg
r-rG per animal) and DT (n=3; equivalent to 250 mg
DT per animal) emulsified in TiterMax in a 1:1 ratio. Primary immunization of
female dogs that comprises of three injections of the respective protein at
monthly interval is underway. Publications Original
peer-reviewed articles 1.
Rath A, Batra D, Kaur R, Vrati S and Gupta SK (2003) Characterization
of immune response in mice to plasmid DNA encoding dog zona pellucida
glycoprotein-3. Vaccine (in press). 2.
Sivapurapu N, Upadhyay A, Hasegawa A, Koyama K and Gupta SK (2003) 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. Mol Reprod Dev (in press). 3.
Govind CK, Srivastava N and Gupta SK (2002) Evaluation of the
immunocontraceptive potential of Escherichia coli expressed recombinant
non-human primate zona pellucida glycoproteins in homologous animal model. Vaccine
21:78-88. 4.
*Rath A, Choudhury S, Hasegawa A, Koyama K and Gupta SK (2002) Antibodies
generated in response to plasmid DNA encoding zona pellucida glycoprotein-B
inhibit in vitro human sperm-egg binding. Mol Rep Dev 62:525-533 (*in
press last year, since published). 5.
*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 56:77-91 (*in press last year, since published). 6.
*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
123:847-857 (*in press last year, since published). 7. *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 47:303-310 (*in press last year, since published). Reviews/Proceedings 1.
Gupta SK (2003) Status of immunodiagnostic and immunocontraceptive vaccines in
India. Adv Biochem Engineer Biotech (in press). 2.
Saxena NC and Gupta SK (2002) Comment on article discussing preimplantation
genetic diagnosis for gender selection for family balancing in India by
Malpani and Malpani. Reprod BioMed Online 4:105. 3. Upadhyay A, Sivapurapu N, Rath A and Gupta SK (2002) Rationale of synthetic peptides as immunogens for vaccine development. Proc Indian Natl Sci Acad B68:97-114. Patents 1.
Jethanandani P, Santhanam R and Gupta SK (1998) Zona pellucida protein, its
preparation and use as infertility agent. Indian patent application no.
1060/Del/98 dated 24 Apr 1998, granted in Sep 2002. |