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.
Bansal
Pankaj
P
Reproductive Cell Biology Laboratory, National Institute of Immunology, New Delhi, India.
Chakrabarti
Kausiki
K
Gupta
Satish K
SK
D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004527:Egg Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013075:Sperm Capacitation / Q000235:genetics; D013084:Sperm-Ovum Interactions / Q000235:genetics; D013094:Spermatozoa / Q000378:metabolism / Q000502:physiology; D000072216:Zona Pellucida Glycoproteins
D018076:DNA, Complementary; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins; C035027:egg surface sperm receptor