Identification of epitopes of monoclonal antibodies to porcine zona pellucida 3 beta glycoprotein, a homologue of the mouse/human sperm receptor.
OBJECTIVE
Immunization with zona pellucida (ZP) glycoproteins leads to a block in fertility with a variable degree of ovarian dysfunction. To avoid autoimmune oophoritis, synthetic peptides corresponding to B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. The main objective of the present study is to define the epitopes recognized by monoclonal antibodies (mAbs) generated against porcine ZP3 beta, a homologue of the designated primary sperm receptor in mice and humans.


METHODS
A multipin synthetic peptides approach has been used to map the epitopes recognized by mAbs. Dodecapeptides with an overlap of 6 amino acids corresponding to a precursor pZP3 beta-deduced amino acid sequence (excluding the signal sequence) were synthesized on polypropylene pins and were tested for their reactivity with mAbs by enzyme-linked immunoadsorbent assay (ELISA). The ability of synthetic peptides corresponding to the identified epitopes to inhibit the binding of mAbs to pZP3 beta in a competitive inhibition ELISA was investigated to confirm the above findings.


RESULTS
Reactivity of the mAbs with the pin-bound peptides in ELISA-identified epitopes for mAb-451 to EEKLVF (166-171) and mAb-462/470 to FKAPRP (250-255) amino acid residues. mAb-30 recognized QPVWQDEGQRLR (23-34) and VICRCC (316-321) amino acid residues. Competitive inhibition with synthetic peptides encompassing the motifs corresponding to 23-34 and 316-321 for binding of mAb-30 to pZP3 beta revealed the epitopic domain to be 23-34 amino acids. Synthesis of overlapping octapeptides further identified WQDE as the minimum motif for binding of mAb-30, and the replacement of one amino acid at a time with glycine revealed tryptophan as the critical residue.


CONCLUSIONS
Collectively, these results describe peptide epitopes that will help in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZP3 beta or its homologues in other species.
Afzalpurkar
A
A
Gamete Antigen Laboratory, National Institute of Immunology, New Delhi, India.
Gupta
S K
SK
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D001402:B-Lymphocytes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D004527:Egg Proteins / Q000276:immunology* / Q000378:metabolism; D018604:Epitope Mapping; D000939:Epitopes / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000276:immunology* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine; D000072216:Zona Pellucida Glycoproteins
D000911:Antibodies, Monoclonal; D004527:Egg Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse; C035027:egg surface sperm receptor