Mapping of epitopes relevant for induction of acrosome reaction on human zona pellucida glycoprotein-4 using monoclonal antibodies

Abstract

PROBLEM: To decipher structural and functional aspects of human zona pellucida glycoprotein-4 (ZP4), the epitopes recognized by monoclonal antibodies (MAbs) have been mapped. METHOD OF STUDY: Recombinant human ZP4-mediated induction of acrosome reaction in human sperm was studied in the absence and presence of ZP4-specific MAbs. The epitopes of MAbs were mapped using recombinant peptides expressed in Escherichia coli. RESULTS: Monoclonal antibodies (MA-1662, MA-1671) against human ZP4 showed specific binding to ZP matrix of human eggs in an indirect immunofluorescence assay. Both the antibodies showed significant (P < 0.05) inhibition in the baculovirus-expressed recombinant ZP4-mediated acrosome reaction. MA-1671 recognized N-terminal fragment of ZP4 and minimal epitope mapped to amino acid residues 126-130 (PARDR), whereas MA-1662 reacted to C-terminal fragment and minimal epitope mapped to amino acid residues 256-260 (ENELV). CONCLUSIONS: The epitopes corresponding to both N- and C-terminal parts of human ZP4 may be relevant for its biological activity.