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.
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.
Govind
C K
CK
Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Hasegawa
A
A
Koyama
K
K
Gupta
S K
SK
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001402:B-Lymphocytes / Q000276:immunology*; D003269:Contraception, Immunologic; D004527:Egg Proteins / Q000276:immunology*; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005260:Female; D006801:Humans; D008255:Macaca radiata; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013084:Sperm-Ovum Interactions / Q000187:drug effects*; D015044:Zona Pellucida / Q000276:immunology*; D000072216:Zona Pellucida Glycoproteins
D000911:Antibodies, Monoclonal; D004527:Egg Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C082833:ZP4 protein, human; D000072216:Zona Pellucida Glycoproteins