Functional activity of human ZP3 primary sperm receptor resides toward its C-terminus

Abstract

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 Nterminal 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 Gi 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.