Identification of epitopes of monoclonal antibodies to porcine zona pellucida 3 β glycoprotein, a homologue of the mouse/human sperm receptor

Abstract

Problem: 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.