Glycosylation of zona pellucida glycoprotein-3 is required for inducing acrosomal exocytosis in the bonnet monkey

Gahlay, G. K. ; Gupta, S. K. (2003) Glycosylation of zona pellucida glycoprotein-3 is required for inducing acrosomal exocytosis in the bonnet monkey Cellular & Molecular Biology (Oxford), 49 (3). pp. 389-97. ISSN 0145-5680

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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.

Item Type:Article
Source:Copyright of this article belongs to C.M.B. Association.
Keywords:Acrosome Reaction; Recombinant Zona Pellucida Glycoprotein; E. coli Expression; Baculovirus Expression; Spermatozoa Binding
ID Code:72134
Deposited On:28 Nov 2011 06:17
Last Modified:28 Nov 2011 06:17

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