Glycosylation of zona pellucida glycoprotein-3 is required for inducing acrosomal exocytosis in the bonnet monkey.
To investigate the role of polypeptide backbone vis-à-vis glycosylation of the putative primary sperm receptor, the bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 (bmZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was expressed as polyhistidine fusion protein either in Escherichia coli orusing baculovirus expression system. The recombinant bmZP3 (r-bmZP3) was purified using nickel-nitrilotriacetic acid resin and subsequently refolded in the presence of oxidized and reduced glutathione. SDS-PAGE and Western blot analysis revealed approximately 43 and approximately 52 kDa bands corresponding to E. coli and baculovirus expressed r-bmZP3, respectively. The r-bmZP3 purified from both E. coli and baculovirus binds to the principal segment of the acrosomal cap of the capacitated bonnet monkey spermatozoa as evaluated by indirect immunofluoresence assay. In a competitive inhibition assay, the binding of biotinylated baculovirus expressed r-bmZP3 to capacitated spermatozoa was inhibited not only by cold baculovirus expressed r-bmZP3 but also by E. coli expressed r-bmZP3 and vice-versa. Lectin binding studies revealed that the baculovirus r-bmZP3 has N-linked glycosylation with galactose and mannose residues. Capacitated spermatozoa, in the presence of baculovirus expressed r-bZP3, undergoes a significant increase (p < 0.01) in the acrosomal exocytosis as compared to control whereas E. coli expressed r-bmZP3 failed to have a significant effect. These results suggest that though the polypeptide backbone of ZP3 is sufficient for its binding to capacitated spermatozoa, yet glycosylation is required for induction of acrosomal exocytosis.
Gahlay
G K
GK
Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 1100 67, India.
Gupta
S K
SK
D000177:Acrosome / Q000378:metabolism*; D020101:Acrosome Reaction; D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D004527:Egg Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005089:Exocytosis; D005260:Female; D006031:Glycosylation; D008255:Macaca radiata; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D018076:DNA, Complementary; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D000072216:Zona Pellucida Glycoproteins