Functional Activity of Human ZP3 Primary Sperm Receptor Resides Toward Its C-Terminus

Zona pellucida glycoprotein 3 (ZP3) has been ascribed as a putative primary sperm receptor during fertilization in humans. Herein, attempts have been made to delineate the functional domain of human ZP3. ZP3 has been cloned and expressed in a baculovirus expression system as N-terminal fragments (amino acid [aa] residues 1-175 [pAc-ZP3((1-175) (aa))]and 23-175 [pBg-ZP3((23-175) (aa))]) and as C-terminal fragments (aa residues 214-305 [pBg-ZP3((214-305 aa))] and 214-348 [pBg-ZP3((214-348 aa))]). ZP3 encompassing both N- and C-terminal fragments corresponding to aa residues 1-370 (pAc-ZP3([1-370 aa])) has also been expressed. Lectin-binding analysis with these recombinant proteins revealed the presence of N- and O-linked glycosylation. Significant induction of acrosomal exocytosis was observed when capacitated sperm were incubated with pBg-ZP3((214-348 aa)), pBg-ZP3((214-305 aa)), and pAc-ZP3((1-370 aa)) (P < 0.05), whereas incubation with pAc-ZP3((1-175 aa)]) and pBg- ZP3((23-175 aa)) failed to do so under similar experimental conditions. However, N- and C-terminal fragments labeled with fluorescein isothiocyanate revealed binding to the anterior head of capacitated human spermatozoa. Escherichia coli-expressed ZP3 C-terminal fragments and chemically deglycosylated pBg- ZP3((214-348 aa)) failed to induce a significant (P < 0.05) increase in acrosomal exocytosis, suggesting the relevance of glycosylation in imparting functional activity to ZP3 C-terminal fragments. pBg-ZP3((214-348 aa))-mediated induction of acrosomal exocytosis is regulated by G(i) protein, extracellular calcium, GABA(A) [gamma aminobutyric acid (A)] receptor-mediated Cl- channel, and T-type voltage-operated calcium channels. Taken together, the results of these studies suggest that the functional activity of human ZP3 resides in its C-terminal domain.