Carboxy-terminal proteolytic processing at a consensus furin cleavage site is a prerequisite event for quail ZPC secretion

• Published in: Biology of reproduction 2003-05, Vol.68 (5), p.1613-1619
• Main Authors: Sasanami, T, Toriyama, M, Mori, M
• Format: Article
• Language: English
• Subjects: active sites; amino acid sequences; Animals; Biological and medical sciences; Blotting, Western; Cell Line; Cells, Cultured; CHO Cells; complementary DNA; Coturnix - metabolism; Cricetinae; cultured cells; Egg Proteins - genetics; Egg Proteins - metabolism; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - ultrastructure; Enzyme Inhibitors - pharmacology; Female; Fundamental and applied biological sciences. Psychology; Furin - antagonists & inhibitors; Furin - chemistry; furin inhibitors; gene expression; glycoproteins; granulosa cells; Granulosa Cells - metabolism; Hydrolysis; immunocytochemistry; immunolocalization; Japanese quails; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Microscopy, Fluorescence; Molecular Sequence Data; mutation; Mutation - genetics; Non mammalian vertebrate reproduction; nucleotide sequences; post-translational modification; protein secretion; proteinase inhibitors; proteinases; proteolysis; proteolytic cleavage; Receptors, Cell Surface; recombinant proteins; RNA, Messenger - biosynthesis; site-directed mutagenesis; Vertebrates: reproduction; zona pellucida; Zona Pellucida Glycoproteins
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod.102.011841

In avian species, a glycoprotein homologous to mammalian ZPC is synthesized in the granulosa cells of developing follicles. We have previously reported that the newly synthesized ZPC (proZPC) in granulosa cells is cleaved at a consensus furin cleavage site to generate mature ZPC prior to secretion. In the present study, we examined the effect of the proteolytic cleavage of proZPC on ZPC secretion by using a specific inhibitor of furin endoprotease and site-directed mutagenesis of the furin cleavage site. Western blot analysis demonstrated that the furin inhibitor efficiently blocked both the proteolytic cleavage of proZPC and the subsequent ZPC secretion. A site-directed mutant that possessed a mutated sequence for furin cleavage was not secreted from the cells. The immunocytochemical observations indicated that proZPC produced in the presence of a furin inhibitor or those produced by the site-directed mutant of the furin cleavage site had accumulated in the endoplasmic reticulum. These results indicate that proZPC is proteolytically cleaved at the consensus furin cleavage site with furin-like protease, and the failure of this cleavage results in its accumulation in the endoplasmic reticulum. Therefore, the C-terminal proteolytic processing of proZPC at the consensus furin cleavage site is a prerequisite event for quail ZPC secretion.