Baculovirus expressed C-terminal fragment of bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 inhibits ZP3-mediated induction of acrosomal exocytosis

• Published in: Molecular reproduction and development Vol. 71; no. 2; pp. 237 - 244
• Main Authors: Gahlay, Gagandeep Kaur, Batra, Deepika, Gupta, Satish K.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.06.2005; Wiley-Liss
• Subjects: Acrosome - physiology; acrosome reaction; Acrosome Reaction - drug effects; Acrosome Reaction - physiology; Animals; Baculoviridae; Biological and medical sciences; Cells, Cultured; Egg Proteins - genetics; Egg Proteins - pharmacology; Exocytosis - drug effects; Exocytosis - physiology; Female; fertilization; Fundamental and applied biological sciences. Psychology; Gene Expression; Hormone metabolism and regulation; Macaca radiata - physiology; Male; Mammalian male genital system; Membrane Glycoproteins - genetics; Membrane Glycoproteins - pharmacology; ovum; Protein Binding - genetics; Protein Binding - physiology; Protein Structure, Tertiary - genetics; Receptors, Cell Surface - genetics; Recombinant Proteins - genetics; Recombinant Proteins - pharmacology; Vertebrates: reproduction; zona pellucida glycoprotein; Zona Pellucida Glycoproteins; Spermatozoa; fertilization; Macaca radiata; Glycoprotein; Monkey; Acrosome reaction; ovum; Germinal cell; Fecundation; Gene expression; In vitro; Zona pellucida; zona pellucida glycoprotein; Virus; Reproduction; Vertebrata; Mammalia; Baculoviridae; Primates; Simioidea; Molecular cloning; Mechanism of action; Oocyte
• ISSN: 1040-452X; 1098-2795
• DOI: 10.1002/mrd.20254

Zona pellucida glycoprotein‐3 (ZP3) has been postulated as the primary sperm receptor in various mammalian species including bonnet monkey (Macaca radiata). However, information on the domain responsible for its binding to spermatozoa is inadequate. In the present study, bonnet monkey ZP3 (bmZP3), corresponding to amino acid (aa) residues 223–348 [bmZP3(223–348)] has been cloned and expressed using baculovirus expression system. SDS–PAGE and Western blot analysis of the purified renatured recombinant protein revealed it as a closely spaced doublet of ∼25 kDa. Lectin‐binding studies documented the presence of both O‐ as well as N‐linked glycans. The biotinylated r‐bmZP3(223–348) binds to the acrosomal region of the capacitated spermatozoa but fails to bind to the acrosome‐reacted spermatozoa as investigated by immunofluorescence studies. In ELISA, nonbiotinylated r‐bmZP3(223–348) and baculovirus expressed r‐bmZP3, devoid of signal sequence and transmembrane‐like domain [r‐bmZP3(23–348)] competitively inhibit its binding to the capacitated spermatozoa. Interestingly, binding of biotinylated r‐bmZP3(23–348) to the capacitated sperm is also inhibited by nonbiotinylated r‐bmZP3(223–348). In contrast to r‐bmZP3(23–348), r‐bmZP3(223–348) failed to induce acrosomal exocytosis in the capacitated sperm. Interestingly, it competitively inhibits the acrosomal exocytosis induced by r‐bmZP3(23–348). These studies, for the first time, identify a domain of ZP3 capable of binding to capacitated spermatozoa and inhibiting ZP3‐mediated induction of acrosomal exocytosis furthering our understanding of mammalian fertilization. Mol. Reprod. Dev. 71: 237–244, 2005. © 2005 Wiley‐Liss, Inc.