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25 February 2009 Functional Activity of Human ZP3 Primary Sperm Receptor Resides Toward Its C-Terminus
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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 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.

Pankaj Bansal, Kausiki Chakrabarti, and Satish K. Gupta "Functional Activity of Human ZP3 Primary Sperm Receptor Resides Toward Its C-Terminus 1," Biology of Reproduction 81(1), (25 February 2009).
Received: 10 November 2008; Accepted: 1 February 2009; Published: 25 February 2009

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